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Copyright N 



COPYRIGHT DEPOSIT. 



A MANUAL 



CLINICAL LABORATORY 
METHODS 



BY 

CLYDE LOTTRIDGE CUMMER, Ph.B., M.D. 

associate professor op clinical pathology, school of medicine, western reserve 

university; associate clinical pathologist, the lakeside hospital; director 

of medicine and visiting physician, st. john's hospital; director 

of laboratories, st. alexis hospital, cleveland, ohio 



ILLUSTRATED WITH 136 ENGRAVINGS AND 8 PLATES 




LEA & FEBIGER 

PHILADELPHIA AND NEW YORK 
1922 






Copyright 

LEA & FEBIGER 

1922 



PRINTED IN U. S. A. 



m 30 ^ 22 

^!.A654418 



TO THE MEMOEY 



MY FATHER 



ROBERT JAMES CUMMER, M.D. 



THIS BOOK IS 



AFFECTIONATELY DEDICATED 



PREFACE. 



In offering this manual for the use of students and practitioners, 
the object is to present clinical laboratory methods in concise and 
accessible form. While devoted largely to a description of methods, 
the underlying principles, the indications for performing tests and the 
significance of the results are outlined. Accordingly the plan adopted 
in most chapters has been as follows: (1) Outline of routine examina- 
tions; (2) description of the simpler qualitative methods which are 
frequently employed; (3) description of quantitative methods or those 
of intricate technic; (4) discussion of findings in various morbid condi- 
tions. When several methods are given, usually the preferred one is 
indicated. 

Details are entered into more fully in some cases than in others. 
For example, the method of counting blood cells has been given 
meticulously, since it is elementary and its procedures are among the 
first which must be mastered by the student. It may seem that 
disproportionate attention has been given to certain subjects, such as 
the histology and pathology of the blood, but experience in teaching 
has shown this to be desirable. 

It is hoped that both the liberal use of cross references in the body 
of the text and the synoptic resumes of quantitative procedures in the 
chapters on Blood and Urine, will facilitate use of the book in the 
clinical laboratory. 

The bibliography, which will be found at the end of the text, contains 
references to original articles on the newer methods, and will prove 
helpful to readers desiring to consult the literature. The standard 
works to which reference is made, have been used freely. 

I desire to express my thanks to many of my Cleveland colleagues 
who read portions of the manuscript and made valuable suggestions, 
particularly to Drs. Henry L. Sanford, Harvey A. Berkes, J. Edgar 
McClelland, Harold N. Cole, Richard Dexter and Carl F. Ulrich and 
to Dr. Henry Schroeder of New York City for his advice regarding the 
relation of urinary examinations to life insurance. I am deeply indebted 



PREFACE 



to Professor Howard T. Karsner of Cleveland for reading and criticiz- 
ing the entire manuscript, and to Professor Victor C. Myers of New 
York City for revising the section on the chemical examination of blood 
and urine. Mr. John. C. Harding of the Cleveland Medical Library 
has rendered invaluable assistance in reading proof, arranging the 
bibliography and verifying references, as did my former secretary, Miss 
Irene F. Higgins, in preparing the manuscript and correcting proof. 

To my wife is due my gratitude for her interest and encouragement, 
which was helpful in the preparation of even this small volume. 

Clyde L. Cummer. 
Cleveland, 1922. 



CONTENTS. 



CHAPTER I. 

The Examination of the Blood. 

Hemoglobin Determination and Enumeration of Corpuscles 17 

Equipment 17 

Indications for Making Blood Examinations 17 

Obtaining Blood for Examination 18 

Determination of Hemoglobin 19 

Comparison of Methods 19 

Sahli's Method 19 

Van Slyke's Gasometric Method 20 

Palmer's Colorimetric Method 22 

Tallquist Hemoglobinometer 24 

The Miescher-Fleischl Hemoglobinometer 24 

The Dare Hemoglobinometer 25 

The Specific Gravity of the Blood 26 

Determination of Specific Gravity of the Blood 26 

Findings from Hemoglobin Determinations 27 

Enumeration of the Red Blood Corpuscles 27 

The Blood-Counting Apparatus 27 

The Enumeration of the Red Blood Cells 33 

Physiological Variations in the Red Count 38 

Variations in Disease 38 

Enumeration of White Blood Cells 38 

Physiological Variations 39 

Enumeration of the Platelets 39 

Wright and Kinnicutt's Method 39 

Method of Buckman and Hallisey 40 

The Color Index 41 

The Volume of the Red Cells 42 

The Volume-index of Capps 43 

The Morphology of the Blood 43 

Preparation of Blood Films 43 

Cover-glass Preparations 43 

Slide Preparations 45 

Staining 47 

General Considerations Regarding Blood Stains 47 

Procedures for Staining with Wright's Stain ... 48 

The Criteria of a Satisfactory Stain 49 

Ehrlich's Triple Stain 50 

The Study of the Stained Preparation 50 

The Differential Count _ 51 

The Findings as Disclosed by Morphological Examination of the Blood 52 

Description of Leukocytes Found in Normal Blood 55 

The Polymorphonuclear Neutrophilic Leukocytes 55 

The Polymorphonuclear Eosinophilic Leukocytes 55 

Polymorphonuclear Basophilic Leukocytes ....... 56 

The Lymphocytes 56 

The Large Mononuclear Leukocytes 57 

The Transitional Leukocytes 57 



vni . CONTENTS 

The Morphology of the Blood— 

Forms of Leukocytes Found in the Blood in Diseased States 58 

Ihe Myeloblasts . . ' co 

The Myelocytes ..*..'.' to 

Turck's " Irritation Forms" . 50 

The Megakaryocytes or Bone-marrow Giant Cells ' ' ' 58 



Cells of Lymphadenoid Origin . 

Ordinary Lymphoblasts ... ' ' ' ' tq 

Pathological Lymphoblasts (Rieder's Cells) '. [ ] 59 



Ordinary Lymphoblasts 
Pathological Lymph 
The Formation of Blood 



The Bone-marrow 



59 



The Origin of the Different Forms of Cell .' ' m 

Life History of the Red Blood Cells ' ' ' «o 

Life History of the White Cells ' ' fi9 

an. ^ ones m , Re g ar d to Blood Formation . ' at 

The Differential Count . . £0 

The Leukocytoses .....'' Jj? 

Pathological Leukocytosis . 'fit 

Lymphocytosis ... ■ ■ . . 00 

The Blood in Childhood and Infancy ! ' ' ' ' fi7 

Special Methods of Examining Blood £0 

Microchemical Blood Reactions '. 'fie 

The Oxydase Reaction . . ' ' " ' «e 

Vital Staining ; °° 

Demonstration of Skeined or Reticulated Staining fiq 

Staimng of Mitochondria in Lymphocytes ' 7n 

Determination of Coagulation Time 4n 

Simple SUde Method . . i" 

Howell's Method .... ' ' ' ' T\ 

Determination of the Bleeding Time 71 

Determination of the Amount of Prothrombin . ' 71 



Resistance of Red Cells to Hypotonic Salt Solution 
Demonstration of Bile Pigments in the Plasma ' 7 q 

The Pathology of the Blood 



The Anemias i^ 

The Primary Anemias, Chlorosis '.'.'.' ' l\ 

Pernicious Anemia ... 7fi 

Aplastic Anemia ...... ''7s 

Secondary Anemias 



79 

79 

Anemias Occurring in Malignancy and the Puerperium ' 7Q 

Anemias Resulting from Infections . . ' 7Q 

Anemia Due to Intoxications . oq 

Anemias Produced by Benzol and Lead Poisoning \ ' 80 
Anemias Due to Poor Hygienic Surroundings ..... .81 

Other^ Anemias Showing Splenomegaly ...... \ fl 



Anemias Due to Hemorrhage 
Chrome Anemias 



Gaucher'^ ^ibcosc 

Banti's Disease ........ 81 

Anemia PseudoleukaBmica Infantum • 0} 

Hemolytic Jaundice 89 

The Leukemias * ' »4 

Chronic Myelogenous Leukemia .... 04 

Chronic Lymphatic Leukemia o? 

Acute Myelogenous Leukemia ....'" ' ' 8? 

Acute Lymphatic Leukemia ... or 
The Clmical Sigmficance of the Findings of Blood Examinations '. 

The Arneth Formula ... so 

Dohle Inclusion Granules .-.'*" ' ' ' on 

Diseases in Which the Blood Contains Parasites ' ' qV 

Msi.ln.nji. yi 

91 

92 



Development of the Parasites 

The Study of the Stained Specimen ' qo 

Infection with Plasmodium Vivax ' 94 



CONTENTS ix 

Diseases in Which the Blood Contains Parasites 
Malaria — 

Infection with Plasmodium Malaria 95 

Infection with Plasmodium Falciparum 95 

Differentiation Between Types of Malarial Infection .... 96 

Summary of the Blood Changes . . . . ' ■ . . ■ . . . . 97 

The Malarial Mosquito 97 

Cultivation of the Plasmodium 98 

Trypansomiasis 99 

Filariasis 99 

Filaria Loa (F. Diurna) 100 

Relapsing Fever 101 

Bacteriology of the Blood 101 

Apparatus 102 

Inoculation of Media 103 

Types of Media 104 

Results of Blood Cultures 106 

Serum Reactions 107 

Agglutination Tests 107 

The Widal Reaction 107 

Isohemagglutinins in Human Serum 112 

Precipitin Reactions for Human Blood 115 

Precipitin Reactions for the Detection of Other Proteins . . . 117 

Complement-fixation Reactions 118 

Principle Underlying Complement-fixation Reactions . 118 

General Consideration and Comparison of Methods 120 

Securing Specimen of Human Blood 122 

Technic of Venipuncture 126 

Collection of Blood in Children 127 

Separation and Inactivation of Serum 128 

Reagents .... 129 

Physiological Saline Solution 129 

Sheep Cell Emulsion _ 129 

Preparation and Titration of Hemolytic Amboceptor . . . 131 

Intravenous Injections 131 

Intraperitoneal Injections 131 

Preparation and Titration of Complement 137 

The Antigens 141 

Controls and Preliminary Set of Reactions 147 

Performance of Reactions Proper 148 

Reading the Reactions 150 

Wassermann Reaction on Spinal Fluid 151 

Interpretation of Results 152 

The Reaction in Treated Cases 154 

Serological Criteria of a Cure in Syphilis 154 

Laboratory Diagnosis of Syphilis of the Central Nervous System . 155 

The Gonococcus Complement-fixation Test 156 

The Antigen 156 

Clinical Value of the Gonococcus Complement-fixation Test . 158 

Complement-fixation Test in Tuberculosis 159 

Craig's Method 159 

Miller and Zinsser's Method 160 

Petroff's Method 160 

Wilson and von Wedel's Method 161 

Clinical Value of Complement-fixation Test in Tuberculosis . 162 

Chemical Examination of the Blood 162 

Routine Procedure 166 

Obtaining Specimen of Blood 167 

Choice of Methods 167 

Determination of Blood Sugar 169 

Methods of Myers and Bailey, Modified from that of Benedict and 

Lewis 169 

Folin and Wu's Method for Sugar Determination 171 

Diastatic Activity of the Blood 175 

Determination of Sugar Tolerance 176 



X CONTENTS 

Chemical Examination of the Blood — 

Blood Urea ^7 

Myers' Method 177 

Folin and Wu's Method ........ . . 180 

Determination of the Non-protein Nitrogen in the Blood . . 183 

Method of Folin and Denis and Greenwald as Modified by Myers 183 

Folin and Wu's Method I85 

Blood Creatinine Igy 

Method of Folin Modified by Myers and Lough ... 187 

Folin and Wu's Method 188 

Determination of Creatinine Plus Creatine in the Blood '. 190 

Folin and Wu's Method 190 

Determination of Uric Acid in the Blood .... 191 

Folin and Wu's Method 191 

Method of Folin and Wu, as Modified by Myers . . . ' ' ' 194 

General Considerations Regarding the Retention of Uric Acid, Urea and 

Creatinine jgg 

Tests for Acid Intoxication 190 

Determination of the Carbon-dioxide Combining Power of the Biood 

Plasma 299 

Method of Van Slyke and Cullen .....'' 199 
Determination of the Reaction to Phenolphthalein of the Protein- 
free Blood-serum 207 



Method of Sellards 



207 



Determination of the Alkali-reserve of the Blood Plasma 90S 

Method of Marriott " 208 

Significance of the Results of Blood Tests for Acidosis .' 211 

Determination of Chlorides of Blood 011 

Method of Myers and Short ... 212 

Austin and Van Slyke's Method .... • • • 

Determination of Oxygen of the Blood '.'.'. 215 

CHAPTER II. 

Examination op Urine 

Routine Examination 017 

Apparatus Required 217 

Daily Amount 218 

Order of Procedure 218 

Appearance of Specimen '.'.'. 218 

Preservation of Specimens 218 

Specific Gravity ... ....... 219 

Qualitative Tests for Protein Bodies .......' 220 

Heat and Acetic Acid Test ..... 220 

Heller's Nitric Acid Ring Test ' 220 

Sulphosalicylic Acid Test 220 

Magnesium Sulphate Test 220 

Record of Albumin Findings 222 

Significance of Albuminuria 222 

Qualitative Tests for Sugar '.'.'.'.'. 223 

Benedict's Test . . 99 o 

Fehling's Test '.'.'.'.' 223 

Fermentation Test 224 

Phenylhydrazine Reaction 904 

Significance of Glycosuria . 225 

Detection of Other Sugars 226 

Microscopic Examination 22fi 

Organized Elements . 997 

r<„„i„ *** 



227 

Mucous Cylindroids ' 2 28 

Significance of Casts in the Urine ...... 228 

Cylindroids .... 99S 

Mucus Threads .'....! '.'.'.'. '...'. 229 



CONTENTS xi 

Routine Examination- 
Microscopic Examination — 
Organized Elements — 

Leukocytes 229 

Red Blood Cells 229 

Epithelial Cells 229 

Yeast Cells 230 

Bacteria 230 

Unorganized Elements 230 

Amorphous Deposits 230 

Sediments in Acid Urines 230 

Uric Acid 231 

Calcium Oxalate 231 

Cystine 231 

Leucine and Tyrosine 231 

Bilirubin or Hematoidin 231 

Hippuric Acid 231 

Sediments in Neutral or Amphoteric Urine . . . . . . 231 

Neutral Calcium Phosphate 231 

Sediments in Alkaline Urine . . . 231 

Ammonium-magnesium Phosphate 231 

Calcium Carbonate 232 

Ammonium Urate 232 

Identification of Inorganic Sediments 232 

Special Qualitative Tests 233 

Tests for Bile Pigments 233 

Foam Test 233 

Gmelin's Test 233 

Troussean's Test 233 

Significance of Bile Pigments 233 

Determination of Bile Acids 233 

Tests for Acetone 234 

Rothera's Test 234 

Gunning's Iodoform Test 234 

Legal's Test .234 

Diacetic Acid 235 

Gerhardt's Test 235 

Test for 5-Oxybutyric Acid 235 

Hart's Test 235 

Significance of Acetone Bodies 235 

Indican 236 

Obermayer's Test 236 

Blood 237 

Benzidine Reaction 237 

Spectroscopic Test 237 

Significance of Hemoglobinuria 238 

Hematoporphyrin 238 

Urobilin 239 

Diazo Reaction 230 

"Bence-Jones Protein" 240 

Nucleoprotein 241 

Quantitative Determinations 242 

Albumin 242 

Esbach's Method 242 

Tsuchiya's Method 242 

Purdy's Method 242 

Sugar 242 

Fermentation Method . ■ 242 

Lohnstein Saccharometer 243 

Reduction Method 243 

Fehling's Quantitative Method 243 

Preparation of Benedict's Reagent 244 

Purdy's Method _ 247 

Quantitative Determination of Glucose by Polariscope .... 248 

Chlorides 249 

Simple Qualitative Test 249 



xu CONTENTS 

Quantitative Determinations- 
Chlorides — 

Harvey's Modification of Volhard's Method . 250 

Determination of Phosphates and Sulphates 251 

Total Nitrogen 251 

Colorimetric Method ... '. o^ 

Urea TT .:::::::::: SS 

Urease ... ... 256 

Determination by Direct Titration of Urine Treated by Urease 256 
Determination by Aspiration and Titration of the Liberated 

Ammonia after Treatment of the Urine with Urease . 256 

Myers' Colorimetric Method 258 

Ammonia 259 

Folin's Method ...... 259 

Folin's Alternate Method ' [ ' 260 

Determination of Uric Acid 251 

Colorimetric Method .... 261 

Determination of Creatine and Creatinine .... 263 

Folin's Later Method, Folin and Morris . . . 263 

Folin's Original Method • • • ^ 

Folin-Benedict and Meyer's Method ..." 264 

Nitrogen Partition ' 2 65 

Chemical Composition of Normal Urine ' 265 

Detection of Mercury in the Urine ' 266 

Tests for Determination of Renal Function . .... 267 

Excretion of Phenolsulphonephthelein as a Test for Renal Function ' 268 

Estimation of the Function of the Separate Kidneys . . 270 

Excretion of Indigocarmin 271 

Mosenthal Test-meal for Renal Function ..." ' 272 

Ambard's and McLean's Coefficients ........ . 273 

Determination of Tolerance to Sodium Bicarbonate as a Test for Acid Intoxi- 
cation 276 

Method of Sellards . . . ' 276 

Bacteriological Examination of Urine ' ' 276 

Examination of Urine for Tubercle Bacilli ' 97A 
The Two- and Three-glass Tests ......[ .'.'..'' 277 

Two-glass Test ' 277 

Three-glass Test 277 

Four-glass Test ' 277 

Examination of Prostatic Fluid '.'.'. 278 

Urinary Findings in Morbid Conditions ......... ' 278 

Acute Nephritis .' ' 279 

Chronic Parenchymatous Nephritis ' 279 

Chronic Interstitial Nephritis '.-'.'.' 279 

Amyloid Kidney ' 279 

Pyelitis 280 

Nephrolithiasis ' 280 

Passive Congestion '.'.'. ' 280 

Cystitis ' 281 



Urethritis 



281 



Diabetes Insipidus 281 

Diabetes Mellitus 281 

Urinalysis with Relation to Life Insurance [ ! 281 



Gastric Contents 



CHAPTER III. 
Examination of Gastric and Duodenal Contents. 



285 



Relative Simplicity of Examination of Gastric Contents ' ' . 285 

Apparatus Required 285 



The Gastric Juice 



285 



Types of Test-meals ..'.'. 287 

Ewald Meal ... . 

Riegel I Meal ] \ [ [ [ \ \ \ 28 7 

Boas Meal 287 



CONTENTS xiii 

Types of Test-meals- 

Dock Meal \ 288 

Salzer Method of Determining Motility 288 

Removal of Gastric Contents ' . ' 288 

Contra-indications to the Use of the Stomach-tube 289 

Obtaining Gastric Contents with Duodenal Tube and the Fractional 

Method of Examination 290 

Routine Examination 291 

Order of Procedure . 291 

Gross Appearance 291 

Qualitative Chemical Tests 293 

Quantitative Chemical Tests 295 

Microscopic Examination 298 

Special Chemical Tests 300 

Pepsin 300 

Qualitative Test for Pepsin 300 

Mett's Quantitative Method 300 

Rose's Method for Quantitative Determination of Pepsin . . .301 

Quantitative Determination of Dissolved Albumin 302 

Volatile Fatty Acids 303 

Sahli's Desmoid Test 304 

Findings in Various Stomach Diseases 304 

Gastric Ulcer ' 304 

Dilatation of the Stomach 304 

Carcinoma of the Stomach 305 

Achylia Gastrica 305 

Continuous Hypersecretion . 305 

Chronic Gastric Catarrh 306 

The Gastric Neuroses 306 

Examination of Duodenal Contents 306 

Obtaining the Duodenal Contents 306 

Normal Appearance of Duodenal Contents . 307 

Tests for Ferments 307 

Findings in Morbid Conditions 308 

Bacteriological Study of the Duodenal Contents 309 

Securing Bile by Duodenal Tube 309 

Bile in Pathological Conditions 310 

Choledochitis 310 

Cholecystitis 310 

Cholelithiasis 310 

CHAPTER IV. 

The Examination of the Feces 

Apparatus Required 311 

Composition of the Feces 311 

The Routine Examination 311 

Method of Transporting Specimen 312 

Macroscopic Examination 312 

Form and Consistency 312 

Odor 313 

Mucus 313 

Blood and Pus 313 

Concretions 313 

Parasites 314 

Curd-like Masses 314 

Chemical Examination 314 

Reaction 314 

Occult Blood 314 

Benzidine Test 315 

Guaiac Test . 315 

Bile Pigments 315 

Corrosive Sublimate Test 315 

Zinc Chloride Test 316 



xiv CONTENTS 

Microscopic Examination 3 16 

Undigested Food Remnants . . ^Ifi 

Red Blood Cells 318 

Leukocytes ' 3^ 



Mucus 



318 



Crystals 3jo 

Search for Parasitic Ova '.'.'.'. 319 

General Bacteriological Examination '..'"' 319 

Detection of Tubercle Bacilli in the Feces ......... 320 

Method of Isolating Tubercle Bacilli from the Feces .320 

Other Bacteria in the Feces 320 

Isolation of Typhoid Bacilli 321 

Schmidt and Strasburger's Test Diet 321 

Findings in the Feces in Diseases 322 

Typhoid Fever 322 

Dysentery 322 

Cholera 302 

Acute Gastro-enteritis 323 

Obstruction of the Common Bile Duct .......... 323 

Pancreatic Disease 323 

Acute Catarrhal Enteritis 323 

Chronic Catarrhal Enteritis '.'.'.' 323 

Mucous Colitis 323 

Intestinal Ulcers 324 

Carcinoma of the Intestine ........... 324 

Intussusception .... 324 

Thrombosis of the Superior Mesenteric Artery . '. [ '. 324 

Chemical Examination of Gall-stones 324 

Intestinal Parasites ] 324 

Method of Search 324 

Examination for Ameba . 325 

Entameba Histolytica ' 326 

Entameba Coli 327 

Trichomonas Intestinalis 327 

Lamblia Intestinalis 32» 

Balantidium Coli '.'.'. 329 

Balantidium Minutum 329 

Trematodes or Flukes '.'.'. 329 

Fasciola Hepatica 329 

Opisthorchis Felineus 330 

Clonorchis Endemicus 330 

Fasciolopsis Buski 330 

Genus Schistosomum 331 

Schistosomum Hematobium 331 

Schistosomum Mansoni 331 

Schistosomum Japonicum 331 

Class Cestoda '.'.'. 331 

Taenia Saginata ' 332 

Taenia Solium 334 

Taenia Echinococcus 334 

Hymenolepis Nana '.'.'.'.'. 335 

Hymenolepis Diminuta . . \ 337 

Dipylidium Caninum 337 

Dibothriocephalus Latus 333 

Class Nematoda . . 338 

Ascaris Lumbricoides 333 

Belascaris Cati . 339 

Oxyuris Vermicularis '.'.'.'. 340 

Anchylostoma Duodenale '. ...... 341. 

Necator Americanus ' 343 

Strongyloides Intestinahs '.'.'.'. 343 

Trichinella Spiralis '.'.'. 345 

Trichuris Trichiura . . . 346 

Preservation and Staining of Flukes and Cestodes . . 347 

Fixation of Flukes 347 

Fixation of Cestodes 347 

Preservation of Ova in Feces . 347 



CONTENTS xv 

CHAPTER V 

The Examination op Sputum. 

Precautions Against Infection ■ 348 

Obtaining the Specimen 348 

Method of Procedure 348 

Order of Procedure 348 

Gross Appearances of the Sputum 349 

Description 349 

Amount 349 

Consistency 349 

Color - . 349 

Odor 349 

Fibrinous Casts 350 

Curschmann's Spirals . • 351 

Dittrich's Plugs 351 

Bits of Necrotic Tissue 351 

Concretions 351 

Parasites 351 

Microscopic Examination 351 

Unstained Specimen 351 

"Heart Failure Cells" 351 

Curschmann's Spirals . . . 352 

Elastic Fibers 352 

Crystals .352 

Blastomycetes 353 

Actinomycosis 355 

Parasites 355 

Examination of Stained Specimens 355 

Impression Method of Making Preparations 355 

Preparing Slides for the Search of Tubercle Bacilli 356 

Staining for Tubercle Bacilli 356 

Ziehl-Neelsen Method 356 

Pappenheim's Method 357 

Much's Granules 357 

Antiformin Method of Preparing Sputum for Examination . . . 357 

Cultivation of B. Tuberculosis 358 

_ Petroff's Method . _ 358 

Staining the Influenza Bacillus 358 

Cultivation of Bacillus Influenzae 359 

Ecker's Method 360 

Avery's Method 360 

Diplococcus Pneumonia? 360 

Determination of Type of Pneumococcus 362 

Culture Media for the Pneumococcus 362 

Blood Agar . 362 

Sterilization of Bile for Testing Solubility of Pneumococci . . 363 

Rapid Cultural Method . . 365 

Determination of Type of Isolated Organism by Agglutination 

Method 365 

Determination of Type by Precipitin Method 366 

Determination of Type III Pneumococcus 367 

Micrococcus Catarrhalis 367 

Streptococcus and Staphylococcus 367 

Bacillus Mucosus Capsulatus 369 

Bacillus Smegmse 369 

Sputum in Disease 369 

Acute Bronchitis 369 

Chronic Bronchitis 369 

Bronchial Asthma 370 

Acute Lobar Pneumonia 370 

Gangrene of the Lung 370 

Fibrinous Bronchitis 370 

Chronic Passive Congestion 370 

Acute Pulmonary Edema 370 

Malignant Disease of the Lung . 370 

Pulmonary Tuberculosis 370 



CONTENTS 



CHAPTER VI. 



Examination of Body Fluids, Exudates, and Miscellaneous Methods. 

Examination of Spinal Fluid 371 

Technic of Spinal Puncture 371 

Routine Examination of the Spinal Fluid 373 

Gross Appearance 373 

Color 373 

Turbidity 374 

Blood 374 

Cytology 374 

. Differential Count 374 

Chemical Examination 374 

Reduction of Fehling's Solution 374 

Globulin ■ 374 

Noguchi Butyric Acid Test 374 

Pandy's Test' . . . . 375 

Ross-Jones Modification of Nonne's Test 375 

Lange Colloidal-gold Reaction 375 

Wassermann Reaction with Spinal Fluid 379 

Bacteriological Examination of the Spinal Fluid 379 

Infection with Micrococcus Intracellularis Meningitidis 380 

Infection with Diplococcus Pneumoniae 380 

Infection with B. Influenzae 380 

Infection with B. Tuberculosis 381 

Infection with Other Organisms 381 

Significance of the Findings 381 

Cell-count 381 

Globulin 382 

Wassermann Reactions 382 

Lange Colloidal-gold Curve . 382 

Tuberculous Meningitis ' 382 

Syphilitic Infections 382 

Acute Anterior Poliomyelitis 382 

Other Infections . . ' 382 

Examination of Thoracic and Abdominal Fluids 383 

Technic of Obtaining Fluid 383 

Gross Characteristics 383 

Specific Gravity 384 

Determination of Amount of Albumin 384 

Cytological Examination 384 

Interpretation of Cytological Findings 385 

Bacteriological Examination 385 

Examination of Purulent Exudates 386 

The Eye 388 

The Ear 389 

The Mouth and Throat 389 

The Nose 392 

Urethra and Vagina 392 

Skin Infections 393 

Achorion Schoenleinii 394 

Ringworm 394 

Trichophytosis Unguis 395 

Microsporon Furfur 395 

Oidium Albicans 395 

Sporotrichosis 395 

Infestation with Itch Mite and Insects 395 

Sarcoptes Scabiei 395 

Pediculus Capitis 396 

Pediculus Vestimenti 396 

Pediculus Inguinalis 396 

Detection of Treponema Pallidum in Syphilitic Lesions 396 



CONTENTS XVI l 

Obtaining Irritation Serum for Examination 397 

Examination with the Dark-field Illuminator i 397 

Giemsa Stain 398 

Wright's Stain 399 

India-ink Method 399 

Detection of Organisms in Soft Chancre 399 

Examination of Semen 399 

Spermatozoa 399 

Florence Reaction 400 

Examination of Brain Tissue for Negri Bodies 400 

Animal Inoculation 401 

Examination of Milk 402 

Human Milk 402 

Dairy Milk 403 

Carbon-dioxide Tension of the Alveolar Air . . 405 

Fredericia's Method 405 

Marriott's Method 407 



CHAPTER VII. 

Bacteriological Methods. 

General Considerations 411 

Direct Microscopic Examination 411 

Cultural Methods 41.1 

Collection of Material 411 

Inoculation of Media 411 

Incubation 412 

Study of Cultures 412 

Isolation of Pure Cultures 413 

Staining Methods _ 414 

Preparation of Media 414 

Preparation of Glassware 415 

Types of Apparatus 415 

Adjustment of Reaction 415 

Colorimetric Determination of Hydrogen-ion Concentration . . . . 416 

Clearing Media 420 

Filtering 421 

Tubing 421 

Sterilization 422 

Hot Air 422 

Steam 422 

Slanting Solid Media 422 

Storing Media .....* 423 

Formulae for Media 423 

Meat Extract Broth 423 

Meat Infusion Broth 423 

Sugar-free Broth 424 

Carbohydrate Media 424 

Dunham's Peptone Solution 424 

Calcium Carbonate Broth 424 

Litmus Milk 424 

Hiss' Inulin Serum-water 424 

Meat Extract Gelatin 425 

Meat Infusion Gelatin 425 

Meat Extract Agar 425 

Meat Infusion Agar 425 

Glucose Agar 426 

Avery's Blood Oleate Agar 426 

Blood Agar 426 

Agar and Broth for Pneumococcus Culture 436 

Hydrocele or Ascitic Fluid Agar 426 

Thomson's Medium for Gonococcus 426 

Petroff s Medium 426 

Loefner's Blood Serum 426 



xviii CONTENTS 

Formulae for Media — 

Dorset's Egg Medium 427 

Potato 427 

Endo's Fuchsin Agar 427 

Russell's Double Sugar Agar 428 

Levine's Simplified Eo^ine-methvlene-blue Agar 428 

MacConkey's Bile-salt Agar 428 

Krumwiede's Brilliant Green Agar 429 

Conradi's Bile Medium 429 

Jackson's Lactose-bile Medium 429 

Robertson's Cooked Meat Medium for Anaerobes 429 



Appendix. 

Examination of a Large Number of Urine Specimens in a Hospital Laboratory 431 

Preparation of Normal Solutions 432 

Preparation of Normal Oxalic Acid 433 

Preparation of Normal Sodium Hydrate 434 

Preparation of Normal Sodium Carbonate 434 

Staining Methods and Preparation of Stains 435 

Loeffler's Methylene Blue Solution 435 

Carbol-fuchsin (Ziehl-Neelsen Solution) 435 

Pappenheim's Decolorizing Fluid 435 

Summary of Technic for Tubercle Staining 436 

Neisser's Stain for the Diphtheria Bacillus 436 

Gram's Stain 436 

Gram's Iodine Solution 437 

Counterstains for Gram's Stain 437 

Summary of Technic for Gram's Stain 437 

Results of Gram's Stain 437 

Capsule Stains 438 

Hiss' Capsule Stain 438 

Welch's Capsule Stain 438 

Staining for Spores 438 

Moeller's Method 439 

Huntoon Method 439 

Staining Flagella 439 

Staining Treponemata 440 

Wright's Stain 440 

Wilson's Stain 440 

Giemsa Stain 441 

Jenna Stain 441 

Antiformin 441 

Liquor Sodse Chlorinatse 442 

Purification of Picric Acid 442 

Preparation of Creatinine Zinc Chloride 443 

Apparatus 444 

Minimum Equipment for Clinical Laboratory 444 

Apparatus Required for Blood Chemistry and Quantitative Determina- 
tions on Urine 445 

Apparatus Required for Serological Tests 447 

Care of Glassware for Serological Work 448 

Preparation of Autogenous Vaccines 449 

Preparation of the Emulsion 449 

Standardization According to a Modified Weight's Method .... 449 

Standardization According to Hopkin's Method 450 

Sterilization of the Vaccine 450 

Dilution and Preservation of Vaccine 451 

Tables of Weights and Measures 451 

Bibliography 454 

Index 465 



CLINICAL LABORATOKY METHODS. 



CHAPTER I. 

THE EXAMINATION OF THE BLOOD. 

HEMOGLOBIN DETERMINATIONS AND ENUMERATION OF 
CORPUSCLES. 

Introductory.— The simplest forms of blood examination are those 
which have to do with the determination of the relative amount of 
hemoglobin, the number of red and white corpuscles, and the study of 
suitably stained films of dried blood. The methods for enumeration 
of corpuscles require practice and patience, but the necessary technic 
is soon acquired when the necessary effort is made. These methods 
should not be attempted at all unless one be willing to spend the time 
to perform them carefully, since the results of hasty work lead only to 
fallacious conclusions. 

Equipment.— The apparatus needed for an examination of this sort 
is relatively simple and is included in the list given in the Appendix 
for the minimum equipment of the clinical laboratory. Apparatus 
and reagents required for unusual procedures will be indicated with 
the description of these methods (Figs. 1 and 2). 

Indications for Making Blood Examinations.— In practically all clinics, 
a white cell count is done routinely together with the hemoglobin 
estimation on admission. If above or in the neighborhood of 10,000 
a stained specimen should be examined, and a differential count made. 
In febrile conditions, in all obscure conditions, and whenever anemia 
is present, recourse should be had to the hemoglobin determination, 
enumeration of white cells, and an examination of a stained specimen, 
with a preliminary differential of at least 150 cells. This does not 
require much time, indeed the more frequently it is done, the easier 
it will be to do, and the information will illuminate many diagnostic 
problems. 

In office practice or in dispensary work a determination of hemo- 
globin should be made almost as a matter of routine. The results may 
either point to the necessity for further examination along hemato- 
logical lines or indicate its futility. A percentage below 85 per cent, 
calls for a count of the red corpuscles, but a figure above this scarcely 
justifies the labor under ordinary circumstances in the absence of 
suggestive clinical signs. 
2 



IS 



EXAMINATION OF THE BLOOD 



Obtaining Blood for Examination.— For Counting Corpuscles, Hemo- 
globin Estimations, etc.— A few drops of blood may be obtained by 
simple puncture of the skin. A lance with a triangular blade is best 
adapted for this purpose (Fig. 2, a), but in case of emergency a clean 
steel pen may be utilized by breaking off one nib (Fig. 2, d). The 




Fig. 1. — Equipment for blood counting, a, Sahli hemoglobinometer; b, Wright's 
stain; c, clean cover-slips; d, blood lancet; e, camel' s-hair brush; /, cover-slip forceps; g, 
hemocytometer (chamber and pipettes); h, distilled water and dropper; z, alcohol lamp; 
j, one ounce bottles for diluents, strapped together with adhesive. 

instrument may be sterilized by dipping the point in alcohol. For 
puncture in adults the fleshy part of the ear lobe or the tip of the 
finger may be used, while with infants and young children it is better 
to use the great toe or the heel. The skin should be prepared by 
rubbing it with alcohol. This is wiped dry and the resulting hyperemia 



CZEQE 



I) 



a. 



b. 



d. 



Fig. 2. — Apparatus used in blood counting, a, blood lancet, preferred form; b, spring 
lancet; c, Stewart cover-slip forceps; d, steel pen which can be used for puncturing 




is allowed to subside. If the lance be handled with the right hand, 
the second finger should be held near the tip, as shown in Fig. 
16, a so that it may serve as a guard against puncturing. After 
puncture the drop should be obtained with the slightest pressure 
possible, made a distance from, rather than close to, the puncture, and 



HEMOGLOBIN DETERMINATIONS 



19 



care should be taken that the drop is not diluted with tissue juices, 
as would be the case if the skin were squeezed. The purchase of 
complicated spring instruments is not desirable (Fig 2, b) . 

Determination of Hemoglobin.— Comparison of Methods.— For general 
utility Sahli's instrument is the method of choice (Fig. 3). When 
carefully standardized, it is reasonably accurate, sufficiently so for 
ordinary clinical purposes. The cost is much less than that of the 
Dare or v. Fleischl instrument. It is available under all circumstances 
since either daylight or artificial light may be employed. Haldane's 
method would be preferable were artificial illuminating gas always 
easily obtained. Both Sahli's and Haldane's 
methods have the advantage of using as the 
standard of comparison a solution of hemo- 
globin derivative. The Sahli method has been 
criticised by numerous workers. It is un- 
doubtedly inaccurate for scientific purposes, 
though for ordinary clinical work it is practical 
and certainly accurate enough, much more so 
than any other readily available method. 
Palmer has devised a method which is both 
simple and accurate, employing carbon mon- 
oxide and the Duboscq colorimeter. The 
disadvantage is the fact that gas is not readily 
available in some localities, the preparation of 
carbon monoxide is not always feasible, and 
the method scarcely lends itself to use except 
when the patient and the laboratory are in 
close proximity. 

Sahli's Method. — First shake the hermeti- 
cally sealed standard tube containing the 
solution of acid hematin. Be sure that the 
graduated tube and pipette are clean and 
thoroughly dry. With a dropper put deci- 
normal hydrochloric acid (y^- HC1) in the 
graduated tube to the mark 10. With the 
pipette draw up blood to the 20 cmm. mark, 
wiping off any blood on the outside of the 

pipette. Eject the blood into the acid in the graduated tube and mix 
by drawing the mixture back and forth in the pipette. Allow this 
to stand one minute, then add distilled water until the color matches 
that in the standard tube, holding both tubes in the black frame 
and viewing them with light transmitted through the ground glass 
background. The height of the fluid in the graduated tube is read 
off on the scale and the percentage of hemoglobin is secured by com- 
puting according to the equation furnished with each instrument. 

The apparatus should be cleaned carefully after use by rinsing the 
graduated tube and pipette with distilled water, alcohol, and ether. 




Fig. 3. — Sahli's hemoglobi- 
nometer. (Simon.) 



20 EXAMINATION OF THE BLOOD 

Before using, be certain that both graduated tube and pipette are 
absolutely clean and dry. 

Renewal of Standard Solution.— -When the standard solution deterior- 
ates, as it will do after a period of months, it should be replaced by fresh 
fluid. We have found the following method a workable one: The 
end of the tube is broken off after making a deep nick with a sharp 
file. The tube is then washed thoroughly with distilled water and 
sterilized by dry heat. Fresh solution is prepared according to the 
following formula : 

Normal human blood 0.5 cc. 

3L hydrochloric acid 5. cc. 

Distilled water 25.0 cc. 

Mix, and add glycerin 25.0 cc. 

Y^- hydrochloric acid may be obtained which will be sufficiently 
accurate for this purpose by adding to 11.7 cc. of HC1 (C.P., U.S.P. 
VIII) sufficient distilled water to bring the volume up to 1000 cc. 

Blood is obtained by puncturing a vein of a normal adult with 
a cell count 5,000,000, using a dry sterile Luer syringe. The yit 
HC1, distilled water and glycerin are sterilized by boiling separately 
in a flask and are cooled before mixing. All glassware should be 
sterilized by dry heat. While the amount of the mixture given above 
is considerably in excess of what is required for filling one standard 
tube, the use of larger quantities is conducive to accuracy. W 7 hen 
the solution has been prepared, the required amount is placed in the 
standard tube, which may be sealed by inserting a small sterile cork, 
dipped in boiling paraffin. 

It is possible to secure new tubes (Eimer & Amend Co., New York 
City, or Steele Glass Co., Philadelphia, Pa.) and to seal them in a 
flame. We have found that it is difficult to obtain tubes of exactly 
the same bore as the calibrated tubes, so it is well to make the standard 
tube furnished with the instrument last as long as possible. 

When working with a standard tube prepared in this way, the per- 
centage may be read directly from the graduated tube after the patient's 
blood has been diluted to match the standard. 

Van Slyke's Gasometric Method for Determining the Hemoglobin.— 
A comparative simple method has been devised by Van Slyke for 
determining accurately the hemoglobin of the blood by means of the 
gasometric determination of the oxygen which it is able to absorb. 
The apparatus which he has described for estimating the carbon 
dioxide content of the plasma is utilized. The other apparatus required 
includes a separatory funnel, glass stirring rod, 2 cc. pipette with its 
lower delivery mark 3 or 4 cm. above the tip, heavy test-tube or 
cylinder, thermometer and barometer. The necessary reagents are 
redistilled caprylic alcohol, ammonia solution made by diluting 4 cc. 
of concentrated ammonia in a liter of distilled water, saponin powder 
if available and saturated solution of potassium ferricyanide. The 



HEMOGLOBIN DETERMINATIONS 21 

latter is freed from oxygen by boiling and is kept in a burette under 
a layer of paraffin oil 2 to 3 cm. thick. 

Procedure.— Three or more cc. of oxalated blood (see page 167) are 
introduced into a separatory funnel and distributed in a thin layer 
about the inner wall to permit the maximum contact with the air 
and to assure complete saturation of hemoglobin with oxygen. The 
funnel is rotated for a few minutes to keep the blood in a thin layer and 
the blood is then transferred to a thick test-tube or cylinder. 

The blood gas apparatus is prepared by introducing into it five drops 
of caprylic alcohol and 6 cc. ammonia solution. If saponin powder be 
available, as much is added to the ammonia as will stick to the end of 
a glass rod. After the ammonia has been introduced into the 50 cc. 
chamber of the apparatus, the chamber is evacuated as described 
elsewhere (page 201) and the air is extracted from the ammonia by 
shaking the apparatus for about fifteen seconds. The extracted air 
is expelled and the process of extraction repeated to make sure that 
no air is left in the solution. Then about 2 cc. of air-free ammonia 
are forced up into the cup of the apparatus. The aerated blood is 
stirred with a rod to secure even distribution of the corpuscles and a 
portion is drawn into the 2 cc. pipette and run under the ammonia in 
the cup of the apparatus. The blood is now drawn into the 50 cc. 
chamber, the ammonia being allowed to follow it and to wash it in. 
An additional drop or two of ammonia may be added from the pipette 
to complete the washing. The blood and ammonia are thoroughly 
mixed in the chamber and allowed to stand until laking is completed. 
When saponin is present, about thirty seconds are required ; when 
not, about five minutes. When complete hemolysis has taken place, 
0.4 cc. of the saturated potassium ferricyanide solution is introduced 
through the cup to set free the combined oxygen. The apparatus is 
now evacuated by lowering the leveling bulb until only a few drops of 
mercury remain above the level of the lower stopcock and shaken with 
a rotary motion to whirl the blood in a thin layer around the wall of 
the chamber. If the blood were completely laked, this should require 
about one-half minute. Since water does not absorb oxygen rapidly 
enough to cause an error, the solution may be left in the 50 cc. chamber 
during the reading and the reading taken by holding at a sufficient 
height to balance the column of the water solution. To make certain 
that all the oxygen was obtained by the first extraction, the apparatus 
is evacuated again, the blood again shaken, and the reading taken 
again. If there is an increase the extraction must be repeated. 

After each analysis the 50 cc. chamber should be washed out with 
the ammonia solution to prevent the formation of a black precipitate. 

Calculation.— First it is necessary to subtract from the gas measured 
the volume of air which is dissolved physically by 2 cc. of blood at the 
prevailing atmospheric pressure and room temperature. The air 
physically dissolved is given in the second column of the accompanying 
table. When the observed reading has been corrected by the sub- 



22 EXAMINATION OF THE BLOOD 

traction of this figure, it is reduced to standard barometric and thermo- 

metric conditions by multiplying by (0.999-0.0046 t) X ba ™™ eter , t 

representing the temperature in degrees Centigrade. Van Slyke's 
table also gives in the third column factors by which the corrected gas 
volume per cent, or in the fourth column the percentage of hemoglobin. 
The percentage of hemoglobin is based upon Haldane's standard of a 
blood having an oxygen carrying capacity of 18.5 per cent., containing 
approximately 14 gm. hemoglobin per 100 cc. 

FACTORS FOR CALCULATING RESULTS FROM ANALYSIS OF 2 CC. OF 
BLOOD SATURATED WITH AIR. 



Temperature. 



Air physically dissolved 
by 2 cc. of blood; sub- . 
tract from gas volume 
read in order to obtain 
corrected gas volume, 
representing O2 set free 
from hemoglobin. 



Factor by which corrected gas volume 
multiplied in order to give: 



Oxygen chemically 

bound by 100 cc. 

of blood. 



Per cent, hemoglobin, 
calculated on the basis 
of 18.5 per cent, oxy- 
gen = 100 per cent, 
hemoglobin. 



C.° 

1.5 

16 
17 
18 
19 

20 
21 
22 
23 
24 

25 
26 
27 
28 
29 

30 



Cc 
0.037 

0.036 
0.036 
0.035 
0.035 

0.034 
0.033 
0.033 
0.032 
0.032 

0.031 
0.030 
0.030 
0.029 
0.029 

0.028 



Cc B 

46.5 X 



Per cent. 



760 



46.3 
46.0 

45.8 
45.6 

45.4 
45.1 
44.9 
44.7 
44.4 

44.2 
44.0 
43.7 
43.5 
43.3 

43.1 



251 X tIo 

2.50 

249 

247 

246 



24.5 
244 
242 
241 
240 

239 
237 
236 
235 
234 



233 



Example of calculation: 

Observed gas volume. Temperature, 20° C, 750 mm. 
Correction for dissolved air (see table above) 
Corrected gas volume, 0.450 minus 0.034. 

0.416 X 44.8 = 18.65 volume per cent, oxygen. 

0.416 X 243.0 = 101.00 per cent, hemoglobin. 



0.450 cc. 
0.034 cc. 



Palmer's Colorimetric Method for the Determination of Hemoglobin.— 
Palmer criticizes the Sahli method because the standard is not perma- 
nent, because there is considerable delay in the development of the full 
color value, and because it cannot be used for the blood of different 
species. He has adopted the use of a standard solution of carbon 
monoxide hemoglobin, preparing a carbon monoxide solution of the 



HEMOGLOBIN DETERMINATIONS 23 

blood to be tested and comparing the unknown with the known in a 
Duboscq or Kober colorimeter. 

The apparatus needed includes a good colorimeter, test-tubes 12 x 120 
mm., a glass stirring rod, and pipettes calibrated to contain 0.05 cc. and 
0.1 cc. The pipettes may be readily prepared from straight glass 
tubing, since they require no blowing. The point may be rounded off 
on an emery wheel. 

The solutions needed are 0.4 per cent, solution of ammonia, made by 
diluting 4 cc. of strong ammonia up to 1 liter with distilled water. 
There must be at hand the standard hemoglobin solution prepared 
from blood having an oxygen capacity of 18.5 per cent. This may be 
determined by Van Slyke's gasometric method (see page 20), or when 
the standard has been properly prepared once, new ones may be made 
up by standardization against it. The blood is diluted with 0.4 per 
cent, ammonia solution to make a 20 per cent, solution of blood having 
an oxygen carrying capacity of 18.5 per cent. This 20 per cent, blood 
solution is saturated by running illuminating gas through it for ten 
minutes. Foaming may be prevented by adding a drop of caprylic 
alcohol. The glass tube through which the gas is passed is withdrawn 
slowly and the bottle stoppered immediately with a paraffined cork. 
Rubber stoppers must not be used in connection with hemoglobin 
solutions. The stopper is sealed with paraffin and kept in the 
refrigerator protected from light. Palmer states that standards so 
made have been kept nearly a year with no deterioration. For use in 
the test a 1 per cent, solution is prepared by adding 95 cc. of 0.4 per cent, 
ammonia solution to 5 cc. of the 20 per cent, blood solution. This 1 
per cent, standard may be made up from time to time and kept in a 
dark glass bottle or in one painted black. Fresh 1 per cent, solutions 
should be made up every two or three weeks. 

Procedure.— Blood is obtained in the usual manner from the finger or 
the lobe of the ear. The flow should be free. A 1 per cent, solution 
is made by drawing 0.05 cc. into the pipette and transferring it to 5 cc. 
of the 0.4 per cent, ammonia solution in a test-tube 12 x 120 mm. 
After the blood has been placed into the ammonia solution, some of the 
mixture is drawn up into the pipette and ejected several times. Ordi- 
nary illuminating gas is allowed to bubble through the solution by 
way of a gas tube for thirty seconds. The solution is now placed in 
the right hand cup of the colorimeter and the standard 1 per cent, 
solution placed in the left hand cup. The standard is set at ten and 
the cup containing the unknown solution is adjusted to it, when the 

reading is taken. The calculation is p + 100 equals the per cent. 

hemoglobin. In the equation, R represents the reading of the scale 
of the right hand cup containing the unknown solution. If the blood 
is markedly anemic, 0.10 cc. should be used and the percentage obtained 
by the calculation divided by two. Venous blood may be used, 
coagulation being prevented by using a crystal of sodium or potassium 
oxalate in the syringe which is used for venipuncture (see page 167). 



24 EXAMINATION OF THE BLOOD 

Saturation of the blood by carbon monoxide should be performed 
under a hood or under a funnel attached to a suction pump. In case 
illuminating gas is not available, carbon monoxide may be generated 
by heating oxalic acid with concentrated sulphuric acid and by passing 
the gas through sodium hydroxide solution to free it from carbon 
dioxide. 

Tallqiiist Hemoglobinometer.— This consists of a book of absorbent 
papers and a scale of colors lithographed on paper, the values ranging 
from 10 to 100 per cent. A drop of blood is absorbed by a bit of 
absorbent paper furnished in the book and, when superficial moisture 
has disappeared, the drop is compared with the scale of colors by mov- 
ing it under the perforations in the color scale. The color which 
matches is taken as indicating the percentage. The absorbed drop of 
blood should be large enough to fill the perforations entirely. The 
method is quite inaccurate, for there is a great variation in the color 
scales. The scale is especially misleading with anemic bloods, and the 
information it furnishes for computing the color index in such cases is 
often worse than useless. 

The Miescher-Fleischl Hemoglobinometer.— This standard apparatus 
gives excellent results but is suitable for laboratory use only on account 
of its size and the fact that a dark room is required for the readings 
(Fig. 4). By drawing blood with a special pipette (Fig. 4, a) to the 
1, f, or § marks dilutions of 1 : 200, 1 : 300, and 1 : 400 respectively 
are possible. The small lines on either side of the marks represent 
twq of the length of the column, so that, when the column of blood 
is not exactly on the desired line, it may be used without adjustment 
by correcting the result accordingly. A dilution of 200 times is too 
strong for normal blood, since the reading cannot be transposed to the 
scale. The blood is diluted with 0.1 per cent, sodium carbonate 
solution rather than distilled water, since the color matches the scale 
better. First one side of the deeper cell (Fig. 4, b) is filled with distilled 
water, and then diluted blood is blown out from the pipette into car- 
bonate solution in the other half of the cell. Each side should show a 
convex meniscus. The glass cover is slid onto the cell carefully so as 
to leave the two chambers exactly full, and the cap (Fig. 4, c) placed 
over the top to hold it in position and limit the field of vision. The cell 
is now placed on the stand (Fig. 4, d) of the instrument. A yellow 
light, such as that from a candle or an oil or gas light, is reflected from 
the mirror (Fig. 4, e) so that both halves of the cell are equally illumi- 
nated. Sunlight or an electric light must not be employed. Readings 
are taken by moving the glass prism with the milled head until the 
color of the prism as seen through the water-filled half of the cell 
exactly matches that of the diluted blood. The eyes should be rested 
during the process, as the retina tires rapidly for red, and the scale 
should be moved by rather wide excursions to both sides, diminishing 
the excursions until the colors are the same. At least 5 or 6 readings 
should be made and the average taken. After the blood has been 



HEMOGLOBIN DETERMINATIONS 



25 



matched in one cell, it may be transferred with the pipette to the cell 
of lesser depth and readings taken in this. The cells are respectively 
15 and 12 mm. high, so that the readings with the lower cell when 
multiplied by f should equal those obtained with the higher. The 
final readings may be translated to mg. hemoglobin per 100 cc. of blood 
by means of a scale which accompanies each instrument. 




Fig. 4. — The Miescher-Fleischl hemoglobinometer. a, pipette; b, cell; c, cap; d, cell 
in position; e, mirror. 



The Dare Hemoglobinometer.— The instrument is comparatively 
costly and easily broken (Fig. 5). There is the additional disadvantage 
that the color scale is not prepared from hemoglobin derivatives (as 
it is with the Sahli instrument), but, like the Miescher-Fleischl, is col- 
ored glass. Undiluted blood is obtained by allowing it to run into 
the receiver, formed by two rectangular pieces of glass which are held 
together in a metal receptacle with a set-screw. The square space 
should be entirely filled with blood, and the receiver is slipped into its 
place in the instrument, when it is viewed against a dark background, 
preferably in a darkened room, with transmitted candlelight through the 
eye-piece. The scale is rotated with the thumb screw until the color 
of the blood and scale matqh, when the percentage may be read from 
the wheel. The instrument has the advantage of a permanent color 
scale, and gives reliable results in the hands of those accustomed to its 
use. Electrically illuminated instruments ase now obtainable, the 
bulb is covered with a shield so that it is not necessary to make 
readings in a dark room. 



2fi 



EXAMINATION OF THE BLOOD 



The Specific Gravity of the Blood.— Some workers employ a determina- 
tion of the specific gravity of the blood as a means of calculating its 
hemoglobin content. 

Determination of Specific Gravity of the Blood.— In a clean and dry 
glass urinometer cylinder prepare a mixture of chloroform and benzol 
in such proportions that the specific gravity will be 1.050 to 1.060. 
Prick the patient's finger and allow a moderate sized drop of blood to 
fall directly into the chloroform-benzol mixture. The drop must not 
be too large, otherwise it will separate and cause confusion. If the 
drop falls to the bottom of the cylinder, it is apparent that the specific 
gravity of the mixture is too low, so chloroform should be added drop 
by drop. When the drop floats on the top, the mixture is too heavy 




Fig. 5. — Dare's hemoglobinometer. 



and benzol should be added. In this case add enough benzol to cause 
the drop to sink to the bottom, then drop in chloroform gradually. 
When the specific gravity of the mixture is the same as that of the 
blood so that the drop is suspended near the center of the liquid 
column, the mixture is filtered and its specific gravity determined by 
hydrometer. 

Hammerschlag's table gives the data require for ascertaining the 
percentage of hemoglobin from the specific gravity. 



Specific gravity. 
1.033 to 1.035 
1.035 to 1.038 
1.038 to 1.040 
1.040 to 1.045 
1.045 to 1.048 
1.048 to 1.050 
1.050 to 1.053 
1.053 to 1.055 
1.055 to 1.057 
1.057 to 1.060 



Hemoglobin. 
25 to 30 per cent. 
30 to 35 " 
35 to 40 
40 to 45 
45 to 55 
55 to 65 
65 to 70 
70 to 75 
75 to 85 
85 to 95 " 



HEMOGLOBIN DETERMINATIONS 27 

Findings from Hemoglobin Determinations.— If 100 per cent, is 
accepted as an arbitrary standard for normal male adults, the per- 
centage with females runs from 90 to 95 per cent. In infancy the 
amount is much higher as has been shown by the recent work of V. B. 
Appleton using Van Slyke's gasometric method. The figures are as 
follow: 

Average, 
Age. per cent. 

1 day . 164 

2 to 3 days 146 

4 to 8 " 135 

9 to 13 " . , ' 137 

2 to 8 weeks 102 

3 to 5 months '. 88 

6 to 11 " 87 

11 to 23 " ' 85 

In childhood the amount usually reaches 75 to 85 per cent, of the 
normal adult standard in the sixth year, and gradually increases until 
the tenth year when it has attained the adult standard. 

The variations due to age and sex have been carefully studied by 
Williamson using the spectrophotometric method of analysis. He 
has shown that the average hemoglobin content of the adult male is 
16.92 gm. per 100 cc. of blood. If this be accepted as representing 100 
per cent, on an arbitrary standard and the Sahli hemoglobinometer be 
standardized accordingly, the findings of Williamson may be translated 
to terms of this standard according to the scale given in the last column 
the diagram (Fig. 6) which has been taken from his work. The dia- 
gram illustrates graphically the variations due to age and sex. 

Enumeration of the Red Blood Corpuscles.— A number of different 
types of counting chambers are upon the market. 1 We recommend 
the Biirker type of chamber with Neubauer ruling and prefer the Levy 
manufacture on account of the clear visibility of the rulings and the 
general type of construction (Fig. 7). Only standardized outfits 
should be employed, and it is more than desirable to secure those whose 
accuracy has been certified to by the United States Bureau of 
Standards. 

The Blood-counting Apparatus.— This consists of two pipettes for 
diluting the blood, a counting chamber, and a perfectly plane cover-slip. 
The pipette used for enumeration of red blood cells has ten marks on 
the stem, the fifth marked 0.5 and the tenth 1. Above the bulb is 
the mark 101 (Fig. 8). When blood is drawn to the 0.5 mark and 
diluting fluid to the 101 mark, the dilution is 1 to 200; while it is 
apparent that with blood drawn to the 1 mark and dilution drawn to 
101 mark, the dilution is 1 to 100. 

The pipette used for enumerating white cells likewise has ten marks 
on the stem, the fifth marked 0.5 and the tenth 1, but above the bulb 
is the mark 11 (Fig. 9). When the blood is drawn to 0.5 mark and 
dilution fluid to 11 mark, the dilution is 1 to 20; while with blood drawn 

1 An excellent historical review of cell-counting technic will be found in an article 
by Horace Gray (Am. Jour. Med. Sci., 1921, clxii. 526). 



2.s 



EXAMINATION OF THE BLOOD 



to the 1 mark and the diluting fluid to the 10 mark, the dilution is 
1 to 10. 



'-o o 

o 6 

£ c5 
J= o 
. o 

i 7 

(3 S. 


1 day 

2 to 3 days 
4 to 8 days 
9 to 13 days 
2 to 8 weeks 
,•3 to 5 months 
6 to 1 2 months 

1 year 

2 years 

3 years 

4 years 

5 years 

6 to 10 years 

1 1 to 1 5 years 
16 to 20 years 
21 to 25 years 
26 to 30 years 
31 to 35 years 
36 to 40 years 
41 to 45 years 
46 to 50 years 
5 Mo 55 years 
56 to 60 years 
61 to 65 years 
66 to 70*years 
71 to 75 years 
76 years and over 




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Fig. 6. — Influence of age and sex on hemoglobin values. Diagram, showing average 
hemoglobin findings at different ages for male and females, according to Williamson's 
findings. 



so sq.mm, 
fe mm. deep. 








MADE BV 

PHILACEIPHIA.PA. 
ArflvurKThisaas Co. 1 





Fig. 7. — Thomas-Levy hemocytometer. 



* This column shows values reduced to a percentage basis if the standard is taken 
as average normal male adult blood, i. e., that containing 16.92 gm. hemoglobin per 
100 cc. 



HEMOGLOBIN DETERMINATIONS 



20 



The Levy chamber consists of a thick glass slide which .is channelled 
transversely with two deep grooves so that there are two parallel walls 
on either side of the rectangular platform (Fig. 10). This platform 



THE ENUMERATION OF THE RED CELLS. 



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Fig. 8. — The enumeration of the red cells. 

Calculation. — The cells in the corner blocks, each made up of 25 small squares, are 
counted on two slides. The total contained in 8 corner blocks as shown in the scheme 
indicated above is 1117. The 8 blocks equal 0.5 square mm., since there are 16 blocks 
of 25 small squares each in a squaremm. Therefore the sum 1117 (contents of 8 blocks) 
is multiplied by 2 to give the contents of 1 square mm., by 10 to give the contents of 
1 cm., since the chamber is only 0.1 mm. deep, and by 200 for the dilution. 

Result.— 1117 X 2 X 10 X 200 = 4,468,000 cells per cm. 



30 



EXAMINATION OF THE BLOOD 



is separated from each of the walls by a depression. The upper sur- 
face of the parallel walls is 0.1 mm. above the upper surface of the 
rectangular platform. The walls have a ground or matt surface, and 
Newton's rings are not demonstrable as with chambers of older types. 



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THE ENUMERATION OF THE WHITE CELLS 









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SCHEME FOR RECORDING RESULTS 
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Fig. 9. — The enumeration of the white cells. 
Calculation. — The cells are counted in the 5 areas outlined by the heavy dotted lines. 
The area of each is 1 square mm. Two preparations should be counted, giving a total 
of 10 square mm. The sum of the contents of the 10 square mm. will give the number 
of cells in 10 square mm., each being 0.1 mm., or in one cubic mm. 

The accurately ground and highly polished cover-glass should bear at 
one corner the word "UP" or the Bureau of Standards stamp. It 
should be mounted so that the side engraved " UP" or the stamp is on 
the upper side. In making a count the cover-glass is placed in position 



HEMOGLOBIN DETERMINATIONS 



31 



on the parallel walls in such a manner that it rests upon them and does 
not touch the slide proper. 

In the older types of instrument the counting chamber consisted of a 
thick glass slide at the center of which is cemented a small circular 




Fig. 10. — Filling the Levy chamber. 



platform (Fig. 11). The surface of the platform is subdivided by small 
rulings, which will be described later. Around the platform is a 
"moat" or "ditch/' separating it from a surrounding "wall" of glass, 
whose inner border is circular and whose outer border is rectangular. 




Fig. 11. — Original type of blood counting chamber, e,, surrounding "wall" of 
glass;/, circular "platform," surface 0.1 mm. below that of surrounding wall; g, 
circular ditch of moat. (Simon.) 



The height of the surrounding "wall" is exactly ^ mm. greater than 
the height of the central platform or disc, so that when the cover-slip 
is placed on the "wall," there is a space between the cover-slip and the 
circular platform just fo ^ nm - deep. 



32 



EXAMIXATIOX OF THE BLOOD 



The simplest form of ruling is found in the Thoma pattern (Fig. 12). 
Here will be found on the surface of the circular platform twenty 
columns running horizontally and twenty running vertically, each 
2V mm. wide. It is evident that when a drop of fluid which has been 
placed on the platform is covered by a cover-slip, the capacity of the 






Fig. 12. — Ruling of Thoma chamber. 



space formed by each tiny square as a base is 3-5-5- cmm. The first 
sixth, eleventh, and sixteenth horizontal and vertical columns are 
subdivided by a median line. This additional line simply serves as a 
guide or landmark. The total area covered by the Thoma ruling is 
1 square mm. 




Fig. 13. — Turck chamber. 



Fig. 14. — Xeubauer chaml cr. 



The Turck and Neubauer rulings are in reality elaborations of the 
Thoma type, the ruled area in both instances covering 9 square mm. 
instead of 1 square mm. The tiny squares of 47^ cmm. area are found 
in the central square millimeter only. The details of these rulings 
can be best understood by reference to the cuts (Figs. 13 and 14). 



HEMOGLOBIN DETERMINATIONS 33 

The Bass ruling (Fig. 15) is extremely simple and is less confusing 
to follow than any of the others since much unnecessary ruling is 
eliminated. Here too the small squares are -^j cmm. square. 



■HI] 
H3H 



Fig. 15. — Bass counting chamber. 

The Enumeration of the Red Blood Cells.— Hayem's solution is a 
convenient diluent since it keeps well and molds do not grow in it with 
readiness. The red cells are readily shaken with it to form a uniform 
suspension, and it is easy to clean the chamber after its use. This is in 
contrast to Toison's solution, much favored by some, for molds grow 
in it readily and the glycerin which it contains renders cleaning 
difficult. The addition of dye to Toison's to stain the white blood 
cells is not an advantage of consequence, since the number of white 
cells encountered in making a red count, even if they were not dif- 
ferentiated, would introduce a noteworthy error in the red count only 
when the white count were exceptionally high. Even Hayem's solu- 
tion, however, should be filtered occasionally to remove possible molds, 
since spores might be mistaken for red cells. 

The formula for Hayem's solution is : 

Mercuric chloride 0.5 gm. 

Sodium chloride 1.0 gm. 

Sodium sulphate 5.0 gm. 

Distilled water 200.0 cc. 

The formula for Toison's solution is : 

Methyl violet, 5B 0.025 gm. 

Sodium chloride 1.000 gm. 

Sodium sulphate 8.000 gm. 

Glycerol . . . . 30.000 cc. 

Distilled water to make 160.000 cc. 

Procedure: Obtaining Blood and Making the Dilution; Precautions.— 
1 . See that the pipette is thoroughly clean and dry and be sure the 
glass bead in the bulb of the pipette may be shaken about freely. 

2. Have ready for use the pipette with the mark 101. 

Obtain a drop of blood according to the previous directions. 

Hold the pipette almost horizontally and with gentle suction draw 
blood to the mark 0.5 (Fig. 16, b) wiping off blood on the outside of the 
3 



34 



EXAMINATION OF THE BLOOD 



pipette with a piece of gauze. If blood is drawn much above the 0.5 
mark, bring it to the 6 mark and correct count by multiplying by f . 
Should the column of blood rise only a slight distance above the 0.5 
mark, draw it back by touching the tip of the pipette with a piece of 






Fig. 1C. — Steps in blood counting, a, puncturing ear; b, obtaining blood with 
pipette; c, drawing up diluting fluid; e, shaking pipette; d and f, preparing pipette for 
transportation. 



HEMOGLOBIN DETERMINATIONS 35 

gauze. Now draw up Hayem's fluid to the mark 101, meanwhile 
rotating the pipette to keep the glass bead in motion, holding it in an 
almost horizontal position (Fig. 16, c). When the pipette has been 
filled, put the thumb over one end of the pipette and the finger over the 
other and shake vigorously (Fig. 16, d and e). 

If it is desired to take the pipette to another place to make the 
count (e. g., from the patient's home to the physician's office), place 
the thumb over the tip of the pipette to seal it, pull the capillary 
rubber tube down to close off the upper end of the pipette (Fig. 16, e) 
and bind the rubber tubing to the upper portion of the pipette by 
means of a small rubber band (Fig. 16, /). The finger now may be 
removed from the tip without any danger of the fluid escaping. This 
will prevent the fluid from running out if the pipette is carried to the 
laboratory in a horizontal position. 

Cleaning the Pipettes'.— Great care must be exercised in cleaning the 
pipettes. Immediately after use the fluid should be ejected. They 
should be filled with water and emptied, then treated successively in 
the same way with alcohol and with ether. The last bit of ether should 
be inhaled, since if it be blown out the moisture of the breath will gain 
access to the pipette. When the cleaning has been well done, the bead 
will be shaken about readily and no moisture will be apparent. The 
cleaning process may be shortened by drawing the various cleaning 
fluids into the pipettes through the tip, and by detaching the rubber 
tubing, connecting it to the tip and then expelling the fluid through the 
larger or upper end of the pipette. The use of a vacuum pump aids 
materially in reducing the labor attendant upon cleaning. Occasionally 
blood coagulates in the pipette, due to slow handling or to drawing up 
alcohol instead of the diluting fluids (a favorite slip with students). 
A bristle or fine wire will have to be run into the lumen with much care, 
and when a passage has been cleared, 10 per cent, sodium or potassium 
hydrate may be drawn in. After standing over night, the pipette 
can usually be cleaned with numerous changes of the same alkali. 

Counting.— When ready to count, clean the chamber and the glass 
cover-slip thoroughly. Never employ xylol, alcohol, ether, or hot 
water on the chamber as both the "wall" and "platform" of the old 
type of chamber are cemented to the glass slide and are readily loosened 
by these solvents, and even in the new chamber the central ruled 
rectangular piece of glass is cemented in. Use only gauze, a clean 
handkerchief or Japanese lens paper with either cold water or the 
moisture of the breath. It is difficult to get rid of bits of lint, unless 
one uses a camel's-hair brush. It is absolutely necessary to have no 
grains of dust, grit or lint on the "wall" or cover-slip. Any foreign 
body in this place, by raising the cover-slip, would increase the distance 
between the cover-slip and the circular platform and so increase the 
number of corpuscles. 

When using the old circular type of chamber, it is the writer's 
practice to clean the slide and cover-slip as directed here and then 



36 EXAMINATION OF THE BLOOD 

place the cover-slip on the slide in the position that it will occupy when 
the preparation is completed. Newton's rings should be looked for. 
They should appear as concentric, vari-colored rings between the 
cover-slip and the "wall" of the chamber when the slightest pressure is 
made on the upper surface of the cover-slip. Their presence indicates 
that the two highly polished surfaces are in perfect apposition. When 
any particles of lint or foreign substance come between the cover-glass 
and the "wall" it will be impossible to demonstrate them. The cover- 
slip is left in place until the pipette has been shaken, when it is lifted 
only long enough to put the drop in place. After shaking the pipette, 
blow out about I of the contents of the pipette onto a piece of gauze 
or blotting paper, and then place a drop on the center of the circular 
platform. 

The size of the drop to be placed on the circular platform can be 
learned only after repeated trials. With the cover-glass in position, 
it must be large enough to cover practically all of the platform, but it 
must not run' into the moat. The cover-glass should be placed in 
position at once. It should not be dropped on carelessly, since in this 
way bubbles will be included, but with one edge resting firmly in a 
diagonal position across the "wall," braced against the thumbs, the 
opposite edge is lowered gently into position with the forefingers. If 
the drop appears to be satisfactory, ascertain whether Newton's rings 
be present. If not, the preparation must be discarded. 

A preparation should not be counted unless it fulfils the following 
requirements : 

1. It should cover all or practically all over the circular platform. 

2. It must not overflow into the moat. 

3. No bubbles should be included in the drop. 

4. Newton's rings must be demonstrated. 

The use of the Levy chamber saves a great deal of time, since the 
construction is such that cleaning is much easier, and the chance for 
the cement between the parts to loosen is not so great as with the old 
circular chamber, the whole slide with the exception of the ruled plat- 
form being of one piece of glass. This reduces the possibilities of 
inaccuracies appearing with deterioration. 

In making a count with the Levy chamber, clean both the slides 
and the ground cover-glass thoroughly, and place the cover in the 
proper position. Shake the pipette vigorously, eject about | of its 
contents, and by holding it in an almost horizontal though slightly 
tilted position, bring the tip to the edge of the rectangular platform so 
that the diluted blood may flow between the platform and the super- 
imposed coverslip (Fig. 10). Stop the flow as soon as the platform 
has been covered and do not allow the fluid to run into the transverse 
channels on either side. 

After preparing a satisfactory drop, it should be allowed to stand for 
a few moments, so that the corpuscles may settle. The area for count- 
ing should be located with IV ocular and the three objective (Leitz), 



HEMOGLOBIN DETERMINATIONS 37 

or 10 ocular and 16 mm. objective (Bausch & Lomb) and then the 
count should be made with the same ocular and with the 6 objective 
(Leitz) or 4 mm. objective (Bausch & Lomb). The microscopic field 
should be darkened by dropping the Abbe condenser slightly and by 
partially closing the iris diaphragm. In this way both the engraved 
lines and the blood cells will be accentuated. Our method of counting 
is to enumerate the number of cells in the four corner blocks, each 
block containing 25 small squares. One such block is contained in 
the field when a magnification of 430 times is used (e. g., 10 eye piece 
and 4 mm. objective of a Bausch & Lomb). This is to be repeated 
on a second preparation. We have found that counting is much 
simplified for students if they prepare a diagram of the blocks to be 
counted and note in each square on the diagram the number of cells 
seen in the corresponding square of the chamber (Fig. 8). The total 
number of cells in each block of 25 squares is ascertained. The total 
of cells in 8 blocks of 25 squares each is multiplied by 2 to find the 
number per square mm., then by 10 to find the number per cubic mm., 
since the chamber is only ^ mm. deep, and finally by 100 or 200 
according to the dilution which was made. 
Example.— First preparation: 

Upper right hand block 139 

Upper left hand block 127 

Lower right hand block 143 

Lower left hand block 136 

Total 545 

Second preparation : 

Upper right hand block ' 146 

Upper left hand block 149 

Lower right hand block 142 

Lower left hand block 135 

Total . 572 

Total for eight blocks of 25 squares each 1,117 

Cells per square millimeter 2,234 

Multiply by 10 for the depth of the chamber 22,340 

Multiply by 200 since blood was drawn to 0.5 mark in pipette 4,468,000 

Criterion for Judging Accuracy in Counting.— The difference between 
the largest and the least number of cells found in two blocks of 25 small 
squares should not exceed 25. A greater difference is evidence that 
the preparations are not evenly spread. In class work, it is our custom 
to insist that students repeat their counts upon their own blood until 
they secure counts upon successive days which check within 300,000. 

Normal Findings.— In healthy male adults the average number of 
red blood cells is usually accepted as being 5,000,000, though it may 
range from 4,500,000 to 5,500,000. For a healthy woman 4,500,000 
is considered normal after the inception of the menstrual cycle. These 
figures are not to be accepted too rigidly. Emerson has shown that 
among medical students the counts ranged from 4,500,000 to 6,700,000. 
Our own experience with large numbers of apparently normal medical 



38 EXAMINATION OF THE BLOOD 

students has been similar. It is not at all unusual to secure counts, 
carefully made and checked, running over 5,000,000 up to 6,000,000. 

In considering counts, the factor of age as well as of sex must be borne 
in mind. In infants immediately after birth the count may run as 
high as 7,000,000, but is usually about 6,740,000 (Stengel & White), 
the average in the nursing period being about 5,580,000 (Wood). 
The count gradually decreases until about the tenth year, reaching a 
minimum of 5,100,000 at or about this time. From that period it 
tends to increase gradually in men until the age of thirty. After 
forty, the count is apt to drop slowly in men and rise in women 
(Emerson). 

Physiological Variations in the Red Count.— Temperature alters the 
count, the count tending to run higher in winter than in summer. A 
count shows a transitory increase after a cold bath. The latter is 
probably due to vasoconstriction. Emerson states that change of 
residence from temperate to tropical regions may cause a diminution 
of from 500,000 to 2,000,000 cells. There is a rise in the count with 
ascent to higher altitudes. 

Variations in Disease.— By oligocythemia is meant a diminution in the 
number of red blood cells per cubic millimeter. It is seen in pernicious 
anemia, chlorosis, the leukemias, the secondary anemias, under which 
heading is included a variety of inciting causes which will be considered 
more fully. 

By polycythemia is meant an increase in the number of red blood 
cells per cubic millimeter. A transient polycythemia is seen after the 
loss of body fluid in sufficient amount to cause concentration of the 
plasma, as may occur in severe diarrhea or persistent vomiting. In 
permanent polycythemias, there is evidently an actual increase in the 
production of new red cells, since the presence of nucleated red blood 
cells and polychromatophilia point to active erythrocyte formation. 
Among the causes of permanent polycythemia are high altitudes, heart 
disease, including mitral valve disease, adherent pericardium, and 
especially congenital heart disease, lung disease, particularly empyema 
and pneumonia, and protracted poisoning stimulating the bone-marrow, 
as by phosphorus and acetanilide. Polycythemia is seen also in a 
disorder whose etiology is undetermined, referred to as erythremia, 
Vaquez's disease, Osier's disease, etc., characterized by great increase 
in the number of red blood cells, and of the total blood volume, by 
cyanosis, retinal changes, and enlargement of the spleen. The 
increased number of red cells is probably due to hyperplasia of the 
erythrogenic tissue of the bone-marrow comparable to the condition 
in myeloid leukemia, where the myelocytic tissue is hyperplastic. 

Enumeration of White Blood Cells.— 0.5 per cent, acetic acid is used 
as a diluent, since this lakes the red blood cells and increases the 
definition of the nuclei of the white cells. Dilute acetic acid may be 
prepared for this purpose by adding 5 drops of glacial acetic acid to 
30 cc. of distilled water. It should be freshly prepared. 



HEMOGLOBIN DETERMINATIONS 39 

The blood is obtained as usual (see page 18). A large drop will be 
needed. Draw this to the mark 0.5 and then fill the pipette to the 
mark 11, with dilute acetic acid. Unusual care will have to be taken 
to make accurate dilutions with the white pipette. The preparation 
on the counting chamber is made in the manner already described 
under "Enumeration of Red Blood Corpuscles." 

This may be done with the IV ocular and the |- objective (Leitz), 
or with the 10 ocular and 16 mm. objective of a Bausch & Lomb, the 
latter giving a magnification of 87 diameters. This magnification is 
well fitted to include the 1 square mm. area. The number of cells 
in an entire square mm. are determined (Fig. 9). In using the Turck, 
Zappert-Ewing or Neubauer rulings, at least 5 square mm. should be 
counted on each of two separate preparations and the average taken. 

Computation.— The average number of cells per square mm. is multi- 
plied by 10, since the chamber is only -^ mm. deep, and then by 20, 
since the dilution was 1 : 20 (Fig. 9) . 

Example.— Suppose that the average number of cells per square 
mm. is 37.25, then multiply by 10 for the depth and 20 for the dilution, 
the result would be 7450 per cmm. 

Normal Findings.— The normal count for adults ranges between 
5000 and 10,000 cells per cubic mm. When the count is below 5000 we 
say that a leukopenia is present, and if above 10,000, there is said to be a 
leukocytosis or hyperleukocytosis. 

In infancy and childhood the count is considerably higher. During 
the first few days of life it may reach 20,000 while in the nursing period, 
the cells average about 12,000. The number steadily diminishes 
during childhood until about the sixth year, when it may range from 
5400 to 12,400 (Wood), conforming to the adult standard at the 
fifteenth year. 

Physiological Variations.— A physiological leukocytosis may be seen 
during digestion, after exercise, during pregnancy, and after cold baths. 
These factors should be borne in mind, and when possible it is well to 
make a blood count before meals. The pathological variations will be 
considered fully after the consideration of the various forms of leuko- 
cytes. 

Enumeration of the Platelets.— Wright and Kinnicutt's Method. — 
Solutions required: 

I. One to 300 aqueous solution of brilliant cresyl blue. This is 
fairly permanent if kept in the refrigerator. 

II. One to 1400 aqueous solution of potassium cyanide. This must 
be freshly prepared. 

Immediately before use, 2 parts of solution I are mixed with 3 parts 
of II, and the resulting mixture should be filtered. 

A very thin cover-glass is necessary. Special ones are made and may 
be obtained from microscope manufacturers in which the body of the 
slip is of usual thickness while the center has been ground out leaving 
a depression with a very thin floor. 



40 EXAMINATION OF THE BLOOD 

Procedure.— Blood should be drawn to the 1.0 mark and stain to 
101 to make a 1 to 100 dilution. 

After filling the pipette, preparations may be made in the chamber 
in the usual way. These should be allowed to stand about ten minutes 
to permit the platelets to settle completely. 

The same method of counting may be employed as in the enumera- 
tion of the red cells. The platelets are seen as small round or elongated 
lilac stained oval bodies. The red cells are decolorized shadows and 
the white cells are easily distinguished because of the dark blue nuclei. 
One hundred small squares may be counted, though where greater 
accuracy is desired, 200 small squares should be counted on each of 
two fillings of the chamber. 

Method of Buckman and Hallisey.— This method has been proposed 
on account of the difficulty in securing brilliant cresyl blue and to make 
possible obtaining specimens of blood in a manner which prevents the 
initial coagulation and consequent thrombin formation with coalesence 
and metamorphosis of platelets. 

The diluting fluid is prepared by dissolving 6 gm. of glucose and 0.4 
gm. of sodium citrate in 100 cc. of distilled water. The resulting fluid 
is filtered and to it is added 0.02 gm. of toluene red, dimethyldiamido- 
toluphenazin ("neutral red"). Solution is slow but eventually com- 
plete. A decigram of crystal violet is added. The mixture is heated 
to 60° C. and held at that temperature for five minutes when it is 
allowed to cool at room temperature. After centrifugalizing for ten 
minutes at 2000 revolutions, the supernatant fluid is filtered twice, 
each time through three thicknesses of dry No. 30 Whatman filter 
paper. The solution keeps indefinitely after the addition of 0.2 cc. 
formaldehyd solution. 

Blood is obtained by venipuncture, using a paraffined 18 gauge 
hypodermic needle, attacked to an evenly paraffined miniature trans- 
fusion tube of the type of Lee and Vincent. This is simply a small 
test-tube open at the top with lower end drawn to a capillary opening 
which fits the needle. The capacity should be about 6 cc. As the 
blood wells up into the tube, the tip of the pipette used for counting red 
cells is introduced and blood is drawn up to the 0.5 mark when the 
pipette is filled immediately with diluting fluid. After shaking three 
minutes the counting chamber (Tiirck or Neubauer) is filled. On this 
preparation, a red count may be made in three minutes, a white count 
in five minutes (count the contents of 9 square millimeters) and the 
platelets in twenty minutes. For the platelets, the high dry objective 
is used and 4 square millimeters are counted. A second preparation 
should be counted and the average taken. 

The authors state the counts may be made as long as four hours after 
taking the specimen. In carefully prepared specimens, the platelets 
are seen as discrete, uniformly distributed, pale blue, ovoid bodies, 
from i to y the size of normal erythrocytes, with finely granular cyto- 
plasm and slightly irregular periphery. The platelet count with this 



HEMOGLOBIN DETERMINATIONS 41 

method is slightly higher than is obtained by other methods. The red 
and white counts check very well. 

Findings.— Wright and Kinnicutt have found that with their method 
the number per cmm. ranges from 224,000 to 330,000, the average 
being 273,250. Normal individuals showed remarkable constancy in 
the counts. 

The findings obtained by Wright agree in a general way with those 
previously obtained by Hayem and also by Pratt. In secondary 
anemia, the count is usually high; in pernicious anemia it is generally 
low. Wright found that in secondary anemia the count was never 
below normal. The highest counts were above 1,000,000 in cases of 
anemia due to hemorrhage. These findings are explained by the 
hyperplasia of the bone-marrow in secondary anemia and the conse- 
quent increase in the megakaryocytes, which produce platelets. Simi- 
larly the dimunition in pernicious anemia is explained by the fact that 
though the red marrow is greater in amount, it does not contain as 
many giant cells as normal. The count was found to be normal in 
two cases of chlorosis. In lymphatic leukemia it is low due presumably 
to the lymphomatous formation of bone-marrow and the consequent 
reduction in giant cells, while in myelocytic leukemia, counts are high, 
ranging from 600,000 to 860,000. 

Early in typhoid fever the counts are normal or below, becoming 
high during convalescence. In lobar pneumonia they are low, normal, 
or slightly below normal, increasing rapidly after the crisis. There 
is no relationship between the platelet count and the leukocyte count. 
Duke feels that the reason for the variation in the count in infectious 
diseases is that small amounts of toxin stimulate and large amounts 
poison the bone-marrow. In two cases of hemophilia, Minot and Lee 
found platelet counts of from 290,000 to 400,000. They advanced 
the theory that the normal number of platelets were unusually resistant 
and therefore unable to liberate a suitable amount of prothrombin. 

The Color Index.— The color index is an expression of the hemoglobin 
content of the red blood cells as compared with the normal. It is 
determined by dividing the percentage of hemoglobin by the per- 
centage or erythrocytes and may be graphically represented by the 
fraction : 

per cent. Hb 
per cent. RBCs 

Of course, the normal color index is one. In computing percentages, 
5,000,000 is taken as the normal red count and 100 as the normal 
hemoglobin percentage. 

Example.— The color index for a patient whose red count is 4,000,000 
and whose hemoglobin percentage was 60 per cent, would be derived 
as follows: 

Four million red blood cells is 80 per cent, of normal. 

The hemoglobin is 60 per cent, of normal. 

The color index is 

per cent. Hb 6C 



per cent: RBCs 80 



0.75 



42 



EXAMINATION OF THE BLOOD 



' The color index is of much practical importance in the consideration 
of, the anemias. 

The Volume of the Red Cells.— By the use of a rapid centrifuge and of 
especially prepared pipettes the relative volume of the red cells in a 
given specimen of blood may be determined. The instrument usually 
employed is the hematocrit of Daland (Fig. 17). The apparatus con- 
sists of two arms which constitute a metal frame, the frame being 
placed on the end of the centrifuge spindle at right angles to its axis. 
Each arm of the spindle carries a glass tube, the outer end of the glass 
tube fitting into a cup-like depression in the frame, the bottom of which 
is padded with rubber. The inner ends of the tubes fit into a similar 
surface which is fastened to a spring which serves to hold the tube 
firmly. Each tube is 50 mm. in length and has a lumen, of 0.5 mm. 
The tube is marked off with a scale of 100 divisions. 

To make determinations, the blood is secured by puncture of ear 
lobe or finger in the usual manner and drawn into the tube by using 
a rubber connection and mouth piece exactly as for the hemocytometer. 
The glass tube must be thoroughly cleaned and dry. The operator's 




Fig. 17.— Daland's hematocrit. (Simon.) 



greased finger is placed over the tip of the instrument while the rubber 
mouth piece is slipped off, and the glass tube is placed in its place in the 
frame, adjusting carefully the rubber. The other tube should be filled 
with water and placed in the opposite side. The rubber pads should 
be greased with vaseline. Speed is essential to prevent clotting of the 
blood, so that all preparations should be made in advance. The 
centrifuge is run for three minutes at a speed of about 10,000 revolu- 
tions per minute to ensure complete separation of serum from cells. 
The leukocytes will be found in a thin white layer at the centripetal 
end of the tube and usually occupy about one division of the scale. 
The red cells occupy about one-half of the tube, the proportion being 
slightly greater for normal males and slightly less for females. While 
the method serves to give an approximation of the number of erythro- 
cytes in many cases, it is open to criticism in cases of severe anemia, 
because of the variation in the size of the red cells in the cases, and in 
leukemia, since the large bulk of leukocytes renders an exact reading 
difficult. 

The chief use made of the method at present is in the determination 
of the volume-index of Capps. 



MORPHOLOGY OF THE BLOOD 43 

The Volume-index of Capps.— This method has been devised by Capps 
to determine the average volume of the erythrocyte. A careful count 
of the red cells is made with a standardized hemocytometer. The 
blood volume is determined with the Daland hematocrit. The per- 
centage number of red cells is determined by multiplying the number of 
million of red cells per cmm. by 20, and the percentage volume of the 
red cells is determined by multiplying the reading on the scale of the 
hematocrit by 2. The volume-index is the quotient obtained when the 
percentage volume is divided by the percentage number. For exam- 
ple, suppose that the column in the hematocrit reaches the mark 45. 
Then the percentage volume is 90. The red cells number four million 
so that the percentage number is 80. The volume index is then 90 
divided by 80, which is 1.12. 

Capps has shown that the cell-volume, as expressed by the volume 
index, is increased in pernicious anemia, usually more so than the 
hemoglobin content of the cell. Moderate cases of secondary anemia 
show only slight loss in cell volume, but in severe anemias, the cells 
are quite small. In chlorotics the cell volume is diminished as well as 
the hemoglobin, but is not reduced to such an extent. Capps feels that 
the volume index is of prognostic significance, stating that patients 
with a normal or nearly normal volume index recover quickly, while 
those with a small volume-index respond to treatment slowly. 

THE MORPHOLOGY OF THE BLOOD. 

The study of the structural details of the various forms of blood cells 
affords valuable information. It may be accomplished by the pre- 
paration of extremely thin films of fresh blood on slides or cover-slips, 
which may be dried, fixed, and suitably stained. 

Preparation of Blood Films.— Films may be prepared for staining by 
spreading a drop of blood on a slide or between two cover-slips. A good 
film is absolutely essential for a satisfactory examination and- so much 
may be learned from a stained film that the very considerable amount 
of time which must be spent in acquiring technic is well spent. Slide 
preparations are comparatively simple to make. However, they are 
open to certain serious objections, the chief of which is that when a 
drop is spread over so large an area one must cover a large portion of 
the film to obtain a really representative count, since usually the leuko- 
cytes are grouped at certain parts of the slide, ordinarily at the edges 
and the ends. A reliable count therefore, requires more time than 
does a count made with a cover-slip preparation. 

Cover-glass Preparations.— To obtain good films, the following points 
must be observed faithfully : 

(a) The cover-glasses must be of the proper size and thickness. They 
should be at least 22 mm. square and should be of No. 1 thickness. 
Before using they should be carefully inspected for imperfections and 
slips showing flaws rejected. Smaller cover-slips are too difficult to 



44 



EXAMINATION OF THE BLOOD 



handle and thicker ones will not permit focusing with the average oil- 
immersion lens. 

(b) They must be absolutely clean and dry and free from dust and 
lint. Unless this requirement be met, considerable labor will be spent 
in vain. 

(c) The drop of blood must be of the proper size, which can be learned 
only by experience. 

(d) All operations must be carried out rapidly to avoid coagulation. 

(e) Constant practice is decidedly helpful in retaining the "knack" 
of making acceptable films. 

Cleaning the Cover-slips.— A number of cover-slips can be cleaned by 
immersing them in chemically pure nitric acid in a covered glass dish. 
After standing fifteen minutes, the acid is poured off and the slips are 
covered very cautiously with 95 per cent, alcohol. A glass plate or 
cover should be held over the dish since gas will be evolved due to the 
chemical interaction between the residual nitric acid and the alcohol. 
After five minutes' time, the alcohol is decanted and the covers are 
rinsed in repeated changes of distilled water until acid cannot be detected 
by litmus. The covers may be kept in 95 per cent, alcohol in a 
covered jar until needed when they may be removed, preferably with 
clean, unstained forceps, to be wiped with clean gauze. They should 
not be dried until needed, since dirt and grease will accumulate upon 
them. 




Fig. 18. — Making films on cover-slips. A, securing drop on one slip; B, pulling slips 

apart. 



Making Cover-slip Preparations.— When the worker is ready to make 
films, one cover-slip should be gripped in the wire forceps by the corner 
and placed on the table. The ear or finger should be pricked and a 
drop of blood obtained (Fig. 16, a). Then a second cover-slip is dusted 
with a camel's-hair brush, passed quickly through the flame of an 
alcohol lamp or Bunsen burner to drive off the moisture, picked up 
carefully by the edge, and applied to the drop of blood in such manner 



MORPHOLOGY OF THE BLOOD 45 

that it will pick up the drop, which should be placed in the center of the 
cover-slip (Fig. 18, a). The ear must not be touched with the glass, 
since oil or dirt adhere. The second cover-slip previously placed in the 
forceps is grasped quickly, dusted with the camel's-hair brush, and 
warmed gently in the flame, when the slip bearing the drop of blood is 
dropped on it gently in such manner that the drop of blood is between 
the two cover-slips. The slip bearing the blood should be dropped on 
the first in such manner that the two slips together look like an eight 
pointed star (Fig. 19, a). The drop of blood is allowed to spread until 
the blood just ceases to flow between the slips when the upper one is 
pulled away with a single quick stroke, (Fig. 18, b), keeping the planes 
of the two slips parallel and the line of traction of both hands in 
parallel planes. 

A satisfactory film should be tongue-shaped, should cover at least 
60 -or 75 per cent, of the total area of the cover-slip (Fig. 19, b), and 
should be thin enough to dry almost instantaneously. On examina- 
tion with the low power of the microscope, the cells through the 
greater portion of the film should be well separated, not touching each 
other, and with normal blood the cell outlines should be circular. A 
film occupying only a small portion of the slip (Fig. 19, c) is not accept- 
able, since there are not enough cells for study. 

Preparation of Smears on Slides.— The slides should be thoroughly 
cleaned with soap and water, rinsed in hot water, and finally in alcohol, 
when they may be rubbed dry with a clean piece of gauze or preferably 
a clean old linen or silk handkerchief. 

A drop of blood of moderate size is obtained and the end of a slide is 
touched to the drop in such a way that the drop will occupy a position 
in the middle of one end. A second slide is now placed over the drop 
with one narrow end in apposition with the upper surface of the first 
slide, holding the first slide horizontal and the second at an angle of 30 
to 45 degrees with it (Fig. 20, A) . The blood situated within this acute 
angle should be allowed to spread along the angle by capillarity when 
the upper side should be pushed along the first slide so as to drag the 
drop with it, spreading it on the first slide (Fig. 20, B). The position 
of the fingers in holding the slide is of importance, since the slides must 
be held so that the upper or " spreader" slide may be pushed practically 
to the end of the slide on which the smear is being made, while firm 
support is given. The posture of the hands is shown in the illustration 
Fig. 20. If the angle between the two slides is more acute than is 
specified the film will be too thin and the cells will be spread over too 
great an area. 

With a slide preparation it is practically certain that some areas will 
be fit for study, and the comparative ease of preparation is a distinct 
advantage to the busy practitioner. There is no objection to the 
method in hunting for malarial or other parasites, but it is open to the 
very distinct objection, already voiced, that the leukocytes are unevenly 
distributed. With a satisfactory preparation, the film should occupy 



40 



EXAMINATION OF THE BLOOD 






a. 



b. 




\ 






( 










'gpp£^= 


^E=^ 




m£ 


^^<f £ -/' 


-<•>>. ^V 


, 


z^ 



f. 




3 



h. 



Fig. 19. — Making and staining blood films, a, proper position of two cover-slips 
when drop of blood is between them; b, well-distributed film; c, poorly distributed film; 
d, staining cover-slip, held in forceps; e, mounted on cork; /, floating on stain; g, well- 
distributed film on slide; h, poorly distributed film on slide. 



MORPHOLOGY OF THE BLOOD 



47 



a broad area across the slide (Fig. 19, g) and should not be a mere streak 
down the center of the slide (Fig. 19, h). 

Staining.— Practically the only stains used for clinical work are 
modifications of Romanowsky's. The results obtained by Leishman's, 
Hastings', and Wright's are practically identical. For clinical work, 
Wright's stain is excellent. Methyl alcohol serves as a solvent for 
the stain and as a fixative, so that preliminary fixation by heat is 
unnecessary. There are a multitude of stains recommended for blood 
work, but it is vastly better to employ one satisfactory stain, to learn 
all that one can about it, to become conversant with the possible 
technical errors and so be able both to guard against them intelligently 
and to interpret the findings correctly, than it is to attempt to employ 
two or three stains. The course of wisdom for one who does only a 
limited amount of blood work would be the complete mastery of a 
single good method. 




A. B. 

Fig. 20. — Making films on cover -slips. A, initial position, allowing drop ofjblood to 
spread in acute angle formed by two slides; B, final position_of upper slide — spread the 
drop as a thin film on lower slide. 



General Considerations Regarding Blood Stains.— The stains of the 
Romanowsky group employ a mixture of methylene blue and eosin. 
The specific staining, however, is not due to the methylene blue, but 
rather to an oxidation product, methylene azure. This is formed in the 
manufacture of Wright's stain by treatment with heat at 100° C. in 
the presence- of an alkali (sodium bicarbonate). It is probable that 
several other by-products are formed, including methylene violet. The 
addition of eosin, a dibasic acid, to methylene blue, a base, results in 
the formation of the eosinate of methylene blue. The resulting £tain 
therefore contains a complexity of substances, the eosinate of methylene 
blue, methylene azure, methylene violet, and possibly other dyes. 
Wilson states that his stain which is prepared by oxidation by silver 
nitrate as well as by sodium bicarbonate, contains seven compounds, 
i. e., eosinate of methylene blue, methylene azure, methylene violet, 
the eosinates of thionin, thionolin, and thionol, and methylene orange. 

The preparation of Wright's stain from methylene blue and eosin is 
given elsewhere in this book (Appendix). If desired it may be secured 



48 EXAMINATION OF THE BLOOD 

in powdered form so that it is only necessary to dissolve small quanti- 
ties in methyl alcohol. This is the most practicable way to prepare the 
stain. The fluid so prepared keeps for a considerable period of time 
when the container is tightly corked. The writer has had good working 
preparations which have given good results at the end of a year, adding 
a little methyl alcohol from time to time to compensate for loss by 
evaporation. Making a satisfactory solution in this way is extremely 
simple, and if contamination with acid or water be scrupulously 
avoided, the results are usually very satisfactory. 

The stain as marketed in the liquid form is very much more expen- 
sive and in many instances is none too satisfactory, failing to stain the 
nuclear elements. 

Procedure for Staining.— Precautions.— It is essential that distilled 
water which is used for dilution, the cover-slip forceps, and the pipette 
used for adding the water be absolutely free from acid, since the least 
trace of acid will result in pale, almost colorless, preparations in which 
it is evident that only the eosin has acted. The points of the cover- 
slip forceps should be wiped quite dry. The precautions seem child- 
ishly elementary, but it is extremely difficult to make students respect 
them, with the result that complaints are made about the poor bottle 
of stain which was their lot. Investigation practically always shows 
that the fault is in the lack of adherence to these simple directions. 
Further, water must never be allowed to get into a bottle of stain. If 
the stain is kept in a dropping bottle with a pipette, this pipette must 
be used for undiluted Wright's only and for nothing else. If the stain 
is kept in a bottle with an ordinary stopper, stain should be removed 
from it only with a dry acid-free pipette. 

The film is grasped in the cover-slip forceps, right side up (Fig. 19, d), 
or placed on a small object, such as a cork or upright test-tube 
(Fig. 19, e) . The film is covered with Wright's stain. Staining may be 
carried out by floating the cover-slip on stain in a staining dish, film 
side down. After a lapse of one and a half minutes, during which time 
fixation has been accomplished by the methyl alcohol, acid-free distilled 
water is added until a metallic film or scum appears on the top of the 
mixture. About equal quantities of water and stain are required. 
This stain is now left for about three minutes. When intense staining 
is desired or when the stain is old and acts slowly, it may be left con- 
siderably longer. It is better to err slightly on the side of overstating, 
since the intensity may be reduced by free washing. The film is now 
washed until it is pink, and the result is viewed with the low power lens, 
according to the standard which follows. If it proves satisfactory, 
it is blotted very gently, dried thoroughly by holding in the flame, and 
mounted on a slide in balsam. 

Should one wish to preserve the stained blood for a long period of 
time, the balsam used for mounting should be neutralized. Powdered 
sodium bicarbonate may be added to Canada balsam which has been 
thinned to watery consistency by the addition of xylol. The sodium 



MORPHOLOGY OF THE BLOOD 49 

bicarbonate is stirred in well with a glass rod and set aside in a corked 
wide mouth glass bottle. At the end of twenty-four hours, or when the 
sodium bicarbonate has settled to the bottom of the bottle, the super- 
natant clear balsam may be decanted and filtered through a folded 
filter paper, previously moistened with xylol. 

The Criteria of a Satisfactory Stain.— With a satisfactory stain, the 
red blood cells should be pink or copper colored without any overcasting 
of blue. This does not refer to an occasional red cell with blue tinge, 
for this may be due to polychromatophilia, but rather to the discolora- 
tion noted throughout an entire film or large portion of it. When 
seen, it usually indicates overstaining. If generally blue, the prepara- 
tion is washed again in tap-water until the red cells have assumed the 
proper color. The nuclei of all leukocytes should be dark blue, the 
exact shade varying with the particular form of leukocyte. If they 
are pale blue, the film has not been stained sufficiently. It should be 
thoroughly dried and the staining process repeated. It is usually just 
as well to start anew with a fresh film, leaving the stain for a longer 
period of time. There is of course the possibility that the stain has 
deteriorated or that either it or the wash water has been contaminated 
with acid. 

Requisites for a Satisfactory Stain.— 1. The cytoplasm of the poly- 
morphonuclear neutrophilic leukocytes should show light violet or 
lilac granules against a pinkish background. If the granules are of 
an entirely pink tint, the film has been washed too long. While it 
may be studied in this condition, the eosinophilic and neutrophilic 
granules will have to be differentiated on the size of the granules. 
The slip may be restained, employing of course less vigorous washing. 

2. The cytoplasm of the lymphocytes should be clear robin' s-egg 
blue. Occasionally it is dark blue, so dark that differentiation of the 
cytoplasm from the nucleus is extremely difficult. This is due to over- 
staining. When the defect is seen, the film should be washed again. 
If the defect cannot be remedied in this way, a new film should be 
stained. 

3. The cytoplasm of the large mononuclears varies from very pale 
blue to robin' s-egg blue. Greater intensity of color betokens the error 
of . overstaining already mentioned. 

4. There should be no granules of precipitated stain generally 
scattered over the entire preparation covering all cells and particularly 
noticeable over the red cells. This will give rise to much confusion. 
It is usually due to allowing the staining mixture to stand too long, 
permitting partial evaporation and a precipitation of stain. This 
fault would cause much confusion so that a slide presenting it should 
be discarded and a new one stained with more care. 

5. There should be no stain on the reverse side of the cover-slip. 
When present it may be easily removed by rubbing with a little filter 
paper moistened with alcohol, but before doing this the slip must be 
laid film side down on a flat surface to avoid breaking it. 

4 



50 EXAMINATION OF THE BLOOD 

Promptness in staining is rewarded by the best results. Specimens 
which have been kept unstained for some time require overstaining 
to bring out the leukocytes properly. As a result, the red cells will 
take the basic stain too intensely. This defect may be remedied 
partially by rapidly plunging the stained film in extremely dilute 
acetic acid (1 drop of glacial acetic acid in 500 cc. of distilled water) 
and promptly washing in distilled water. 

Ehrlich's Triple Stain.— At the present time this stain is used very 
little for clinical work, since it is difficult to prepare and use, and does 
not stain malaria parasites or the basophilic granules in mast-cells. 
It is described here for historical reasons only. 

The stain is prepared from saturated aqueous solutions of methyl 
green, acid fuchsin and orange G. The colors must be chemically pure 
and should be added in the order and proportions indicated in the 
formula given by Morris: 

Orange G., saturated solution 13.0 cc. 

Acid fuchsin, saturated solution 7.0 cc. 

Water 15.0 cc. 

Methyl green, saturated solution 17.5 cc. 

Alcohol 10.0 cc. 

Glycerol 10.0 cc. 

The solutions are measured in the same graduated cylinder, which 
should be rinsed between fluids, and the constituents placed in a flask 
which is shaken after each addition. The mixture should be neither 
shaken nor filtered. Blood films must be fixed by heat. A long 
triangular piece of copper about f inch thick is supported on an iron 
tripod and the pointed end heated with a Bunsen burner about fifteen 
minutes until it has reached a constant temperature. The boiling-point 
is determined by dropping water on the surface and a point selected 
about an inch further from the flame than the place where drops assume 
a spheroid shape. The smear is placed on the plate film side down. 
Periods of time ranging from two minutes to an hour have been 
recommended. 

Fixed films are stained from three to twenty minutes. There is 
little danger of overstaining. The films are washed in water, dried, 
and mounted in the usual way. With this stain, red blood cells are 
buff colored, the nuclei of the leukocytes green, the neutrophile granules 
lilac, the eosinophile granules crimson, while normoblastic nuclei are 
almost black. 

The Study of the Stained Preparation.— The film should be studied 
first with the low objective. This is necessary for a general survey, 
and should always precede a more minute examination. Many hints 
are obtained with this long distance perspective, as it were, which would 
be lost if a close-up view alone were depended upon. The distribution, 
the relative number of red and white cells, and the apparent scarcity 
of red cells may be readily seen in this way. Then the high dry 
objective should be used. If a system giving a magnification of about 



MORPHOLOGY OF THE BLOOD 51 

500 be employed, it is ordinarily unnecessary to employ the oil-immer- 
sion lens for routine counts unless it is desired to work out certain finer 
details, such as the morphology of malarial parasites. 

In making a study, it is desirable to observe the points which follow. 
In submitting reports negative as well as positive findings always should 
be given in detail so that any one who reads the report may have a 
word-picture of the film. For one's own satisfaction it is desirable to 
follow a definite routine. 

Points to be Noted in Routine Study of Stained Specimens of Blood.— 
1. Distribution of the Red Cells.— Are they well and uniformly dis- 
tributed through the body of the slide? 

2. Apparent Number of Red Cells. — Roughly speaking, are as many 
jeen per field as is customary? 

3. Apparent Number of White C elk.— Again speaking approximately, 
are as many seen per field as is customary? It will be well for the 
student or beginner to determine the average number of leukocytes 
in each field of a film of the average thickness when examined with a 
magnification of 500 times. 

4. Relative Proportion of White Blood Cells to Red Blood Cells.— 
Are they apparently normal, increased or diminished ? 

5. Minute Study of the Red Cells, including the following points: 
(a) Is the central pale area normal, increased or diminished? 

(6) Are the red cells of uniform size or is there anisocytosis? 

(c) Are the red cells of normal shape, or is there evidence of poikilo- 
cytosis? 

(d) Have the red cells stained uniformly or is there evidence of 
polychromatophilia ? 

(e) Is punctate basophilia seen? 

(/) Are nucleated red cells seen? If so, what forms? They should 
be counted while one makes the differential count, but should not be 
included with leukocytes. For example, in reporting, one would say 
that in counting 300 leukocytes, 5 nucleated red cells were encount- 
ered; 2 were normoblasts and 3 were megaloblasts. 

6. Blood Platelets.— Are they apparently present in normal numbers, 
increased, or diminished? 

7. Parasites.— If present, detailed study should be made, and an 
exact diagnosis made. Failing in the latter, detailed drawings should 
be made so that they may be submitted to others for opinion together 
with the slide. 

The Differential Count.— At the very least 250 white cells should be 
enumerated. If it be not the intention to cover the entire film in the 
count, a definite system should be followed so some areas will be taken 
and studied in all portions of the film in order that the count may be 
representative. 

A mechanical stage is of great assistance, but as an expedient one 
may use a slide clamped to the stage firmly with a microscope clip. 
The slide bearing the blood film may be pushed along this as a guide 



52 EXAMINATION OF THE BLOOD 

until one column across the film has been studied. Then the guiding 
slide may be moved sufficiently to bring a new field into view and the 
film may be moved back. The process may be repeated until the 
required number of cells has been seen. 

In making a differential count, it is useful to employ the following 
form which follows the classification at present favored in practically 
all clinics. In reporting to others, the total number of leukocytes 
counted should be stated, as well as the proportion of each form. 

Polymorphonuclears, neutrophilic (P.M.N.) 

Lymphocytes (large and small) (L.) 

Polymorphonuclears, eosinophilic (E.) 

Polymorphonuclears, basophilic (mast-cells) (B.) 

Transitionals (T.) 

Myelocytes, non-granular (My.-ng.) 

Myelocytes, neutrophilic (My.-n.) 

Myelocytes, eosinophilic (My.-e.) 

Myelocytes, basophilic (My.-b.) 

Nucleated red blood cells are not leukocytes and while it is desirable 
to note them, if any are seen, of course they are not enumerated as 
leukocytes in the differential. 

When specimens are mounted in balsam, one occasionally sees a 
refractile circle which appears to be within the red cells and is con- 
centric with the cells. In many cases this is due to inclusion of 
moisture resultant upon insufficient drying of the cover-slip. The 
cover-slip and the slide with a drop of balsam should be warmed gently 
over the flame before they are brought together. 

The Findings as Disclosed by Morphological Examination of the Blood.— 
The Red Blood Cells.— The red blood cells are small discoid, possibly 
cup-shaped bodies. The diameter of the normal adult cell averages 
about 7/jl, though it may vary from 6 to 9/z. Cells of normal char- 
acteristics are referred to as normocytes. In infancy a consider- 
ably greater variance is seen, with a large number of much smaller 
cells. By microcytes we mean small cells less than 6/x in diam- 
eter, and by macrocytes over 9/x. in diameter, the term megalocyte 
ordinarily being reserved for a cell over 16ju in diameter. Detection 
of unusual cells is of importance in the diagnosis of the anemias. 
The normal red cell contains no nucleus. Anisocytosis is a term used 
to describe marked irregularity in size. In the fresh preparations the 
cells have a slight greenish yellow tint. After fixation, they manifest 
an affinity for acid stains (acidophilic, oxyphilic), and therefore when 
treated with stains of the Romanowsky group take a pinkish copper 
color, due to the action of eosin (they are eosinophilic) . Normally the 
stained cells show practically uniform depth of color since the hemo- 
globin content is approximately the same. 

Polychromatophilia (polychromasia) is seen in some abnormal cells. 
Because of degeneration or immaturity, the eosin has not been taken 
up well and the basic stain has been partially absorbed. Cells so 
stained are seen in pernicious anemias, occasionally in the leukemias, 



MORPHOLOGY OF THE BLOOD 53 

after certain poisons, in malaria and in certain other conditions. Such 
cells were viewed, especially by Ehrlich, as degenerated cells, but 
present-day opinion is inclined to regard them as immature forms. 

"Cabot's ring bodies" are faint ring-like structures seen with the red 
cells, staining a reddish or bluish tint with the Wright's stain. Cabot 
considered them to be nuclear remnants. Their presence in the 
circulating blood might be construed as evidence of the escape into the 
circulation of only partially matured cells. 

Howell's Nuclear Particles.— These are bits of nuclear material, too 
large to be called granules, having the shape and appearance of a 
nucleolus, usually lying at the periphery of the cell. Howell's explana- 
tion is that they are bits of the nucleus left adhering to the corpuscle 
at the time the nucleus escaped. The particles have been seen in 
experimental anemia in rabbits poisoned by pyrodin, the blood of cats 
following severe hemorrhage, and in pernicious anemia in man. The 
bodies are looked upon as signs of regeneration of the blood. They are 
also referred to as Howell- Jolly bodies. 

Punctate basophilia (punctate basophilia of Grawitz) refers to a 
condition occasionally noted in red blood cells where granules of varying 
size and numbers may be seen, stained with the basic blue stain. The 
granules may be exceedingly small, so small as to be scarcely visible, 
while on the other hand, the size may be considerable. They are 
regarded by some as nuclear remnants, though this is contested by 
Simon, since they do not stain with methyl green, a specific nuclear 
dye. At any rate their occurence, with extremely rare exceptions, 
is evidence of severe anemia. They are seen in pernicious anemia, lead 
poisoning and malaria, occasionally in the leukemias and cachexias. 
Simon states that they are absent in chlorosis and the anemia of chronic 
nephritis. Their detection in suspected cases of lead poisoning is of 
prime diagnostic import. Basophilic stippling may be demonstrated 
with Wright's stain. The films should be stained immediately. 

Shape.— The outline of the red cells as seen in stained preparations 
is circular or practically so. Marked deformity of the cell outline is 
spoken of as poikilocytosis. This is seen best in pernicious anemia. 
There may be variety of irregularities, so that we may see cells of oval, 
pear, tennis-racket, or dumb-bell shape. Before deciding that poikilo- 
cytosis be present, assurance should be had that the irregularity of 
cellular contour be not a mere artefact due to faults in the preparation 
of the film. With artefacts, the long axes of the elongated red cells 
are usually parallel. When poikilocytosis is actually present, it is 
customary to find normal cells in the same microscopic fields, while 
when mechanical injury is responsible entire fields of bizarre deformed 
cells will be found. 

Nucleated Red Blood Cells.— In normal adult circulating blood 
nucleated red cells are never seen. They are found in fetal blood and 
as would be expected, are occasionally encountered in the blood of 
normal infants during the first few days of life. Since they are in the 



54 EXAMINATION OF THE BLOOD 

process of development, they are found in great numbers in the bone- 
marrow. 

In the blood of adults with certain diseases we see various types of 
nucleated red cells. The normoblast is a cell corresponding in size 
to the normocyte, the diameter ranging from 6 to 9ju with a nucleus 
occupying a more or less central position while the protoplasm fre- 
quently shows a tendency to polychromatophilia, (Plate I, a) so that 
the color ranges from red overcast with blue to a greenish-blue. The 
nucleus is usually densely stained with the blue nuclear stain and little 
chromatin network is to be distinguished (pycnotic nucleus), though 
occasionally a radial arrangement of the chromatin may be made out. 
Mitotic figures are occasionally seen (karyokinesis). 

Normoblasts appear in the circulation as a result of hyperactivity 
on the part of the bone-marrow. They may be seen in any of the 
anemias when the marrow is attempting to replace blood rapidly and 
so turns out immature forms, or in diseases of the bone-marrow, such 
as splenomyelogenous leukemia, where all sorts of immature red and 
white cells are discharged into the circulation. When constantly 
absent from the bone-marrow in cases whose blood picture is otherwise 
typical of pernicious anemia, this fact in itself points to lack of any 
endeavor on the part of the bone-marrow to regenerate blood so that 
complex corresponds to an aplastic anemia, the prognosis of which is 
distinctly poor even as regards a favorable remission. 

Mcgaloblasts.— These are nucleated red cells whose diameter ranges 
from 10 to 12ju up to two or three times that of a normoblast, but 
whose nucleus is usually of quite different character, in that it is 
large, is usually eccentric, and ordinarily stains more faintly, having a 
vesicular appearance quite in contrast to the pycnotic type common in 
normoblasts (Plate I, b). The nucleus may show many irregularities 
including vacuolation and subdivisions into small basophilic staining 
fragments. The cell outline is frequently irregular, sometimes almost 
polygonal, even appearing indented in ragged fashion. With cells of 
this type mitotic division of the nucleus is noted at times (Plate I, d) 
and here also we encounter polychromatophilic cytoplasm and punctate 
basophilia. The worker must be on the alert not to dismiss these cells 
as large mononuclear leukocytes, particularly as "Tiirck's irritation 
forms," which will be described later. 

Megaloblasts escape into the circulating blood comparatively rarely 
and are interpreted as signs of an exceedingly grave anemia and of an 
arrest in the natural development of red cells. Broadly speaking they 
are seen in adults only in pernicious anemia or severe anemias of this 
type and in the leukemias; though rare cases of secondary anemia or 
even chlorosis are reported in which they have been seen. In the 
blood of young children their presence does not bring the same serious 
message as with adults, since the blood-forming organs in childhood 
responds with greater changes to much lesser stimuli. 

Microblasts are extremely small red cells, the diameter being not over 
3. 5m, with nuclei similar to those of normoblasts. 



PLATE I 






••V 



o 10 eo 

In 1 1 f . i..l . i . ■ In nT 



C.L. Cummer, micra 

Types of Abnormal Red Blood Cells. 

All cells drawn to same scale as Plates II and III for purpose of comparison. Each 
division of the scale at the bottom of the plate represents one micron. Magnification, 1150. 

Stained with Wright's stain, a, normoblasts; b, erythrocytes showing punctate baso- 
philia; c, megaloblasts; d, megaloblast with nucleus undergoing division. 



MORPHOLOGY OF THE BLOOD 55 

Platelets .—In stained specimens the platelets appear as tiny oval or 
circular bodies about 3/x in diameter, the main substance having 
tinted blue surrounded by tiny reddish granules. They are often in 
little clusters or clumps, entangled with threads of fibrin (Plate II, 14) • 
While their origin is not definitely settled, general acceptance is given 
to Wright's explanation, that they are pinched off from the megakary- 
ocytes, or bone-marrow giant cells. Their function is in connection 
with the coagulation of the blood, evidently the furnishing of pro- 
thrombin. The period of their existence is extremely short, about 
three days. From a practical point of view, it is extremely important 
to learn to recognize these little bodies, since they frequently lie over a 
red cell and are mistaken all too often for nuclei or malarial para- 
sites. 

Description of Leukocytes Found in Normal Blood.— The Polymorpho- 
nuclear Neutrophilic Leukocytes.— Commonly spoken of as "polys.," 
(abbreviated by many as P.M.N.) these cells are the most numerous 
in the circulating blood, being encountered 6 or 7 times out of every 
10 leukocytes which are seen. As observed in stained specimens, they 
are approximately circular, though outline is oval at times. In fresh 
specimens, ameboid movement may be seen. The diameter is about 
9 to 12^, that is, from about one and a half times up to almost twice 
that of the average red cell. The nucleus is quite irregular in form, 
hence the name. It may be U-shaped, S-shaped, multilobular, or 
apparently broken into several fragments which, while apparently 
distinct, are in reality joined by a fine thread of nuclear material 
(Plate I, 1-5 inch). Arneth has classified cells according to the divi- 
sions of the nucleus (see page 89) . The nucleus always takes a basic 
stain, that is, it stains a blue which is not as deep a shade as that of 
the lymphocyte nucleus. Nucleoli are not seen. 

The protoplasm is comparatively abundant. The ground substance 
is somewhat acidophilic, tinted pink. This is not always easy to dis- 
cern since the protoplasm is studded with many fine granules which are 
neutrophilic and as a result stain a lilac color or not infrequently a 
reddish tint due, so Barker states, to the acidophilic property of the 
granules in older cells. Inclusion bodies within the cytoplasm of the 
cells have been described by Dohle. It is to be remembered that the 
description of the staining properties is based upon the results of 
Wright's stain. 

The polymorphonuclear neutrophiles are the so-called "pus cells," 
though when seen in pus they have frequently undergone degeneration. 
Their function is to attack and engulf bacteria and to produce a 
proteolytic ferment. This ferment acts only in alkaline medium and 
its action is inhibited by blood serum, so that in the presence of blood 
serum, its proteolytic activity is carried on only within the cell. It is 
thought that they participate in the formation of complement. 

The Polymorphonuclear Eosinophilic Leukocytes.— The cells are com- 
monly referred to as " eosinophiles." The name is abbreviated to 



56 EXAMINATION OF THE BLOOD 

P.M.E. or E. They are cells similar in size and shape to the neutro- 
philes just described, though possibly slightly larger. Their number is 
much less, one ordinarily encountering only from 2 to 4 in counting 100 
leukocytes, 5 being the upper limit of normal. The nucleus is more 
regular, less broken up, and stains less intensely than that of a neutro- 
phile, frequently so feebly that it is difficult to make out. There are 
no nucleoli. (Pate II, 16, 17.) The protoplasm cannot be seen usually 
on account of large and uniform granules staining a bright intense red 
whose size and refractility renders them easily distinguishable even in 
unstained specimens of fresh blood. It is important to become familiar 
with the comparatively great size of these granules. "When once 
mentally photographed, they should never be confounded with neutro- 
philic granules even if the latter have been stained a red tint rather 
than lilac. Barker's demonstration that the granules contain iron is 
of distinct interest. 

Polymorphonuclear Basophilic Leukocytes.— Commonly these cells are 
referred to as mast-cells. The abbreviations P.M.B. or B. may be 
used. They are comparatively infrequent in normal blood, running 
usually about 0.5 per cent. In size they are smaller than the other 
polymorphonuclear cells, the average diameter being about 8 to 10/x. 
The nucleus, which occupies about two-thirds of the cell, may be quite 
difficult to differentiate since it stains poorly and is frequently eclipsed 
by the deep staining granules which may overlie it. The polymorphism 
of the nucleus is different from that of the two forms of cells just 
described, for it often has rather a knobby, knurled appearance, so 
that it has been described as representing the outline of a clover leaf or 
rosette. 

The granules are of large size, indeed are practically of the same as 
the granules of the eosinophils. They take the basic blue stain very 
intensely, manifesting an almost purple color. The protoplasm is not 
nearly so thickly studded with granules as with the eosinophils, the 
granules are irregular in size, and the protoplasm between them 
appears almost colorless, occasionally faintly red or a delicate blue. 
(Plate II, 21.) 

The Lymphocytes.— ~Fov convenience, the abbreviation L. will be 
used. The proportion in normal adult blood ranges from 20 to 25 per 
cent. The size of these cells runs from 7 to 10//. They contain a 
proportionately large nucleus, practically filling the cell, so that only a 
narrow rim of cytoplasm may be seen about it. This is a variable 
feature, however, and at times a relatively larger amount of cell sub- 
stance may be present. Ordinarily the nucleus has a decided affinity 
for the basic stain so that its color is deep blue, staining much more 
deeply than the nuclei of any of the other forms of leukocytes. Occa- 
sionally a framework may be made out within the nucleus. In each 
nucleus one or two nucleoli are present, small, oval bodies staining blue. 
The protoplasm is not granular. It is feebly basophilic and in satis- 
factory stains is transparent and of faint sky-blue tint. At times the 





PLATE II 



f 3 








% # 



18 
C.L. Cummer 



19 



10 20 

limlmilnnliml 



Found in Normal Blood. 




Types of Cells 

All cells drawn with the same magnification and outlined with the camera lucida for 
the purpose of comparing sizes. Each division of the scale at the bottom of the plate 
represents one micron. Magnification, 1150. 

Stained with Wright's stain. Nos. 1 to 5, inclusive, polymorphonuclear neutrophilic 
leukocytes; 6, 7 and 8, small mononuclear leukocytes; 9, 10, large mononuclear leukocytes; 
11, 12, 13, transitional leukocytes; 14, a group of platelets; 15, a group of red blood cells; 
16, 17, polymorphonuclear eosinophilic leukocytes; 18, 19, 20, "basket cells," degenerated 
leukocytes; 21, "mast-cell," basophilic leukocyte. 



MORPHOLOGY OF THE BLOOD 57 

color will be a little darker at the cell border than close to the nucleus 
(Plate II, 6, 7 and 8). With Wright's stain a few "azure granules" 
are seen, granules of variable size and staining red. This description 
applies to the small lymphocytes or the small mononuclears. 

Under the heading of lymphocytes we must also include the large 
lymphocytes, which are identical in all respects except for greater size, 
the proportion between cytoplasm and nucleus being the same. These 
cells are relatively, sometimes absolutely, increased in typhoid fever 
and other infections. Apparently they are of distinct value in combat- 
ing these infections, since it has been shown by Murphy and Ellis' 
work that animals deprived of lymphocytes by away treatment are 
more susceptible to tuberculosis. On these grounds, it is assumed that 
the round-cell (lymphocyte) infiltration seen as a late stage of an 
inflammatory reaction is an index of the power of the lymphocytes to 
ward off injuries. The work of Webb and Williams is interesting, since 
they have shown that normal adults and children as well as guinea-pigs 
and rabbits living at high altitudes (Colorado Springs) show a high 
mononuclear count as compared with counts made at sea-level. 

The Large Mononuclear Leukocytes. — These are dubbed "large 
monos." The abbreviation L.M. will be used. This form varies in 
size from 10 or 12/x up to 20/x. About 1 to 5 per cent, of all the leuko- 
cytes are large mononuclears. The protoplasm is conspicuously abun- 
dant, in comparison with that of the lymphocytes, occupying a distinct 
area about the nucleus, which is usually eccentrically placed, again in 
contradistinction to the centrally placed nucleus of the lymphocyte. 
While with ordinary staining the protoplasm has practically the same 
staining properties as has that of the lymphocytes, appearing as sky- 
blue and almost transparent, it is uniform throughout, and shows no 
deepening at the cell boundary. "Azure granules" are frequently 
seen. Again by way of contrast, the nucleus does not stain nearly as 
deeply as the lymphocyte nucleus. (Plate II, 9 and 10.) At least a 
large proportion of these cells are phagocytic and contain a proteolytic 
enzyme which acts only in acid medium. They probably exert a 
protective function in tuberculous processes. 

The Transitional Leukocytes .—The transitionals to which we will 
give the abbreviation T. are of large size, exceeding all the other forms 
in this respect. They compose about 2 to 3 per cent, of the total 
leukocytes. The characteristic features are their size and more 
particularly the presence of an indented or notched nucleus. The 
nuclei stain a moderately faint blue about as deep as the nucleus of the 
large mononuclears. The cytoplasm is faintly basophilic taking a 
light blue color, and contains a varying number of lilac-colored neu- 
trophilic granules, similar in appearance to the granules of the poly- 
morphonuclear neutrophiles and often most marked about the notch. 
(Plate II, 11, 12 and 13.) It is now felt that they are a mature form 
of large mononuclears, and should be grouped with the latter in finally 
interpreting a differential count. Their name is a misnomer since at 



58 EXAMINATION OF THE BLOOD 

present they are not considered as transitional forms between large 
mononuclears and polymorphonuclear neutrophiles. 

Forms of Leukocytes Found in the Blood in Diseased States.— The 
Myeloblasts.— These are occasionally called non-granular myelocytes. 
In development they are the precursors of the granular myelocytes, 
which are in turn the forerunners of the granulated polymorphonuclear 
cells of normal circulating blood. The size is variable. Small forms 
are occasionally seen, but ordinarily the diameter is from 12 to 20;u. 
The nucleus is sometimes round, more frequently oval, stains a light 
blue, and contains a number of nucleoli. The practically clear proto- 
plasm stains very light blue. These cells are found in the bone-marrow 
and may escape into the circulation only in certain pathological condi- 
tions. (Plate III, 4, 5 and 6.) 

The Myelocytes.— The myelocytes are normally present in the bone- 
marrow and are found in the circulating blood only in pathological 
conditions. They are large in size, the diameter ranging from 15 to" 
20/x or more. The nucleus stains a rather faint blue, and contains 
nucleoli. Usually it is not in the center of the cell. The surrounding 
cell substance is conspicuous by its large amount, is slightly basophilic 
(blue) and studded with granules, which may be eosinophilic (staining 
red), basophilic (staining blue) or neutrophilic (faint reddish-blue or 
lilac staining). Accordingly the myelocytes are classified respectively 
as eosinophilic myelocytes, basophilic myelocytes, or neutrophilic 
myelocytes. With the first two forms, the granules are large, while 
the granules of the latter are small, corresponding in size to the granules 
of the similar granulated polymorphonuclear cells of normal blood 
which are descended from them. They arise from myeloblasts of the 
bone-marrow. An interesting variant is an occasional cell containing 
both eosinophilic and basophilic granules. (Barker attributes this 
to the youth of the granules.) Usually in leukemic bloods the neu- 
trophilic myelocytes predominate. While the myelocytes often are 
divided into these classes in executing a differential count, it must be 
admitted that no especially useful purpose is served bv so doing. 
(Plate III, 1, 2, 3, 7, 8, 9, 10.) 

We must remember that all of these cells are merely stages in the 
process of development of the polymorphonuclear cell of normal blood 
from the myeloblasts and that many transitional forms will be en- 
countered in leukemic bloods. 

Tiirck's "Irritation Forms."— This is a large mononuclear cell 
resembling the myeloblasts in size, shape, and in the character of the 
nucleus. The protoplasm, however, stains a deep reddish-blue and is 
not infrequently vacuolated. It is considered by some to be a patho- 
logical myeloblast and is seen in the circulation in severe infections, 
in severe anemias and in myelogenous leukemia. 

The Megakaryocytes or Bone-marrow Giant Cells.— These cells seldom 
escape into the circulation. They are of great size and have large and 
convoluted nuclei. Occasionally multiple nuclei are seen. 



p >," 



PLATE III 



2 







C.L. Cummer 



• » 



O 10 ZO 

I 1 I 1 1 I I I I 1 1 I I I I I I I I T I 



IE 



Leukocytes Found in Circulating Blood Under Abnormal 
Conditions. (Stained with Wright's Stain.) 

All cells drawn with camera lucida with the sarne magnification and to the same 
scale as Plates I and II for the purpose of comparison. Each division of the scale at the 
bottom of the plate represents one micron. Magnification, 1150. 

Nos. 1, 2 and 3, neutrophilic myelocytes; 4, 5 and 6, immature myelocytes, non-granu- 
lar, with basophilic myelocytes; 11 and 12, lymphoblasts. 



MORPHOLOGY OF THE BLOOD 59 

Cells of Lymphadenoid Origin. — The cells just described (myeloblasts, 
myelocytes, Tiirck's "irritation forms" and megakaryocytes) are of 
myeloid of bone-marrow origin. Under other abnormal circumstances 
we may see cells discharged into the circulation which have come from 
the lymphatic tissue. 

Ordinary Lymyhoblasts .— These are essentially similar to the ordinary 
lymphocytes, though somewhat larger in size and possessed of more 
protoplasm. They have their origin in the germinal centers. They 
may be seen in the blood of normal children but in adults' blood only 
under pathological conditions. (Plate III, 11 and 12.) 

Pathological Lymphoblasts (Rieder's Cells).— These are lymphoblasts 
of large size, seen occasionally in acute lymphatic leukemia, at times 
in the chronic form, in various infectious diseases and Basedow's disease. 
The nucleus is large, and there is little protoplasm, the nucleus often 
coming to the edge of the cell on one side. "Azure granules" may 
be seen in the cytoplasm. (Plate III, 11 and 12.) 

The Formation of Blood.— Introductory.— Before entering into a con- 
sideration of the anemias and leukemias, it is necessary to review the 
formation of the blood cells and their life-history. In the embryo the 
primitive cells apparently are formed from the endothelial cells of the 
capillaries. Later the majority of the cells in the fetal circulation are 
hemoglobin-bearers, while the leukocytes are present in small numbers. 
Practically all the hemoglobin-bearing cells contain nuclei and are 
produced in various organs, particularly the liver. About the third 
month of fetal life, the formation of bone-marrow begins and this 
gradually supplants all other organs in the production of all cells except 
those of lymphocytic origin. The bone-marrow produces both large 
and small erythroblasts and later non-nucleated red blood cells. The 
polymorphonuclear cells appear comparatively late. At the time of 
birth, the blood contains non-nucleated red cells, neutrophilic leuko- 
cytes, lymphocytes, and a few normoblasts, neutrophilic myelocytes 
and myeloblasts. Eosinophilic and basophilic leukocytes are rare. 
Soon after birth the normoblasts, myeloblasts and myelocytes disappear 
from the circulation to reappear only under abnormal conditions. 

In the adult we have under normal conditions as blood-forming 
(hematopoietic) organs probably only the bone-marrow and the 
lymphoid tissue. It is possible that under abnormal conditions, when 
the bone-marrow is unable to produce the red cells in sufficient numbers, 
a certain amount of red cell formation occurs in the spleen. In myelog- 
enous leukemia the liver, spleen and other organs may contain deposits 
of myelocytes, which are presumed to be production centers for these 
cells. 1 

The Bone-marrow . — An understanding of the formation of the blood 
is greatly facilitated by a study of the bone-marrow. As was stated, 
this does not begin to function until the third or fourth month of fetal 

1 For a discussion of the pathological physiology of blood-cell formation and blood- 
cell destruction, the student is advised to consult the article by Cecil K. Drinker in 
The Oxford Medicine, Oxford University Press, New York and London, 1920, ii, 509. 



60 EXAMINATION OF THE BLOOD 

life, after which time it is quite active. At birth the shafts of the long 
bones are filled with red marrow, but during the period of childhood 
this is gradually replaced by fatty tissue, leaving only small deposits 
of actual marrow in the diaphyses of the long bones, in the bodies of 
the vertebra and the cartilaginous ends of the ribs. When severe 
anemia occurs and there is a need for replacement of blood or in 
myeloid leukemia, the fatty marrow may be displaced by actively 
proliferating marrow, containing red and white cells and their precursors. 
For the purpose of study, a portion of the bone from the middle of 
the shaft of the tibia should be obtained at autopsy, as soon as possible 
after death. It should be cut into with bone forceps and a little of the 
marrow squeezed out on a cover-slip, mixed with a droplet of physio- 
logical salt solution and carefully spread as directed for the preparation 
of blood smears with cover-slip method. The resulting film should be 
stained with Wright's stain and carefully studied. In it will be found 
all the cells which are found in normal circulating blood, since it is 
needless to say that this has access to the bone-marrow, and in addition, the 
cells found in the marrow only. Therefore the following should be seen : 

1. Ked cells in all stages of development, normoblasts with one or 
more nuclei, an occasional megaloblast, and the normal red cells which 
we see in circulating blood. 

2. White cells as seen in circulating blood, polymorphonuclear cells 
with neutrophilic, eosinophilic, and basophilic granules, lymphocytes, 
large mononuclears, and transitional cells. 

3. White cells which are seen in the circulation only in disease but 
are normally present in bone-marrow, giant marrow-cells (megakaryo- 
cytes), myeloblasts, myelocytes with neutrophilic, basophilic and 
eosinophilic granules and other border-line forms which may be difficult 
to classify. 

The Origin of the Different Forms of Cell.— The polymorphonuclear 
cells, regardless of the type of granulation, have their origin in the bone- 
marrow and are derived from neutrophilic myelocytes which in turn 
are derived from the non-granular myeloblasts. As has been stated 
all these forms, and hence all stages of development, may be seen in 
preparations from bone-marrow. The genealogy of a polymorpho- 
nuclear neutrophilic leukocyte is myeloblast, neutrophilic myelocyte, 
neutrophilic polymorphonuclear leukocyte (Fig. 21). 

Many large mononuclear cells likewise have their origin in myeloid 
tissue, and transitional cells are now presumed to be a later stage. 
Some of the large mononuclear cells probably arise from the endothelial 
cells. of the capillaries, since oxydase granules cannot be demonstrated 
in all of these cells. While there can be little doubt that a certain 
proportion of the large mononuclear cells come from the endothelial 
cells of the capillaries, it is equally certain that certain of the large 
mononuclears are of myeloid origin because they contain oxydase 
granules. Therefore it is not fitting to apply the term endothelial 
leukocytes to all the large mononuclears, but rather to retain the 
generally accepted terminology here given, remembering that it 



MORPHOLOGY OF THE BLOOD 



61 



includes two types of cells having similar appearance but different 
sources. The transitionals are not, as their misleading name would 




62 



EXAMINATION OF THE BLOOD 



imply, a transitional stage previous to development into polymor- 
phonuclear cells. 

The lymphocytes are of lymphadenoid origin, arising from lymph- 
glands, splenic Malpighian follicles, and lymph nodules. 

Life History of the Red Blood Cells.— It is of interest to follow the 
history of the red cells. Their duration of life was estimated by 
Quincke as one month. The work of Ashby, tracing the life of trans- 
fused blood in the circulation of the recipient by means of agglutination, 
shows that their life is for thirty days or more. The birthplace is the 
bone-marrow and their "graveyard" is chiefly the spleen, where dam- 
aged or dying cells are caught by the macrophages. This function 
is performed to a lesser extent by bone-marrow and liver and is taken 
over by the lymph nodes after splenectomy, as has been shown by 
Pearce. After destruction the non-iron bearing pigment is carried to 
the liver, where it is transformed to biliary pigments and is so excreted 
via the bile ducts and intestinal tract. 

Life History of the White Cells.— The tenure of life of a leukocyte is 
unknown. Many of course are lost in combating infection and by 
way of various secretions (mouth, nose, throat, etc.). In leukemia the 
destruction of leukocytes is tremendous and because of the breaking 
down of nucleoproteins, the endogenous uric acid is greatly increased, 
as is nitrogen-metabolism in general. 



TABLE SHOWING FORMS OF ERYTHROCYTES, LEUKOCYTES, PLACES OF 
ORIGIN, AND CONDITIONS OF PRESENCE IN CIRCULATING BLOOD. 











Ever present 






Present in 


Present in 


in circulating 


Form of cells. 


Origin. 


bone- 


normal circu- 


blood in path- 






marrow? 


lating blood? 


ological con- 
dition? 


Erythrocytes. 










Normocyte 


Bone-marrow 


Yes 


Yes 


Yes 


Normoblast 


Bone-marrow 


Y^es 


No 


Yes 


Megaloblast 


Bone-marrow 


Yes 


No 


Y'es 


Leukocytes. 










Polymorphonuclear neutrophiles 


Bone-marrow 


Yes 


Yes 


Yes 


Polymorphonuclear eosinophiles 


Bone-marrow 


Yes 


Y^es 


Yes 


Polymorphonuclear basophiles 


Bone-marrow 


Yes 


Yes 


Y r es 


Mononuclears, large 


Bone-marrow 


Y^es 


Yes 


Y^es 


"Transitional" cells 


Bone-marrow 


Yes 


Y r es 


Yes 


Mononuclears, small 


Lymphoid 
tissue 


Yes 


Yes 


Yes 


Ordinary lymphoblasts 


Lymphoid 
tissue 


Yes 


No 1 


Yes 


Pathological lymphoblasts 


Lymphoid 


Yes? 


No 


Yes 


(Rjeder's cells) .... 


tissue 








Megakarocytes 


Bone-marrow 


Yes 


No 


Seldom 


Myeloblasts 


Bone-marrow 


Yes 


No 


Yes 


Tiirck's "irritation-cells" . 


Bone-marrow 


Yes 


No 


Yes 


Myelocytes, basophilic . 


Bone-marrow 


Yes 


No 


Yes 


Myelocytes, neutrophilic . 


Bone-marrow 


Y^es 


No 


Yes 


Myelocytes, eosinophilic . 


Bone-marrow 


Yes 


No 


Yes 



Occasionally, in'childhood only. 



THE DIFFERENTIAL COUNT 63 

Theories in Regard to Blood Formation.— It has been noted that we 
have assumed a double origin for the blood cells, the formation of a 
portion in the bone marrow and a portion in the lymphadenoid tissue, 
that in each instance all cells formed in a given tissue have had a com- 
mon ancestor, but that these two common ancestor-cells were quite 
distinct. It should be stated that while this theory of Ehrlich's has 
been adopted by many hematologists it is not universally supported 
and that there is a considerable school which follows the lead of Pappen- 
heim in regarding all leukocytes as descended from a common ancestor 
cell. In our opinion very strong evidence in support of the theory of 
Ehrlich is the demonstration of oxydase granules in the cells of bone- 
marrow origin and their absence in the lymphocytes. Furthermore it 
would seem that the theory of Pappenheim, going a step further than 
Ehrlich's, requires much additional proof which has not been forth- 
coming in convincing form. 



THE LIFE HISTORY OF THE RED BLOOD CELLS. 



Place of origin. 


Duration of life. 


Function. 


Place of 
destruction. 


Disposal of end- 
products. 


Red blood cells 


One month or 
more 


Oxygen-bearing 


Spleen chiefly; 
also liver and 
bone-marrow 


Non-iron-bearing 
pigment carried 
to liver, here 
transformed to 
biliary pig- 
ments, excreted 
as bile via intes- 
tinal tract. 


Leukocytes 


Unknown 


Combating in- 
. fection; pro- 
teolytic action, 
etc. 


In blood stream 


Nitrogen from nu- 
cleo-protein ex- 
creted by urine. 




In exudates 


Lost in secretions 
from mouth, 
nose, throat, etc. 



THE DIFFERENTIAL COUNT. 



The directions for making a differential count of the leukocytes have 
been given in a preceding section. For study of the data so secured, 
the all-important requisite is a normal standard. The standards long 
given have impressed many as not at all approximating the average as 
obtained in counts on apparently normal subjects. 

The wide variation in standards set down by different workers is 
well illustrated by the table given on page 64. 



64 



EXAMINATION OF THE BLOOD 





-a_ 










1 1 ! 










Form. 




■3 

H 

1 


i 


>> 


O 


tnon. 

merson. 

ebster. 


c 


J 


1 


1 
1 




a 


X 


u 


1 


£ 


x K £ 


W 


S 


H 


e» 


P. M. N. 






















Per cent. 


70-72 


60-74 2 62- 


70-72 


65-75 


60-70 70-72 65-75 


50-60 


64.2 


60-70 


62.0 


P. M. E. 






















Per cent. 


2-4 


1-5 


0.5-4 


2-4 


0.6-11 


1-4 2-4 2-4 


0.8-1.0 


2.8 


1-4 


1.0 


P. M. B. 






















Percent. 
L. 
Per cent. 


0.5 


1 


./u-Vso 


0.5 


0.5 


0.5-1 


0.5 0.5 


0.4-4.0 


0.6 


0.5-1 


0.2 


22-25 


25 


20-30 


22-25 


22-25 


20-30 


22-25 20-25 


30-40 


22.4 


25-33 


28.0 


L. M. 
























Per cent. 
T. 
Per cent. 


1 


5-8 


4-85 


2^6 


1 


1-6 


1 3-5 


0-6-2.0 


8.0 


2-5 


7.5 


1-3 


4 


4 


7 


2-4 


' 13 ' 


6.0-8.0 


2.0 


4 





It would seem that the most extensive work in this country has been 
done by Miller, who has performed 650 counts on 230 normal persons. 
The figures which he takes as representing an average are given in the 
table above, and by Sondern, who performed 500 counts. Combining 
their figures, we should suggest the following as a normal standard : 



Polymorphonuclear neutrophiles 

Lymphocytes 

Large mononuclears and transitionals . 
Polymorphonuclear eosinopliiles 
Polymorphonuclear basophiles (mast-cells) 



62.0 to 64.0 per cent. 
22.0 to 28.0 

7.5 to 10.0 

1.0 to 3.0 

0.2 to 0.6 



The Leukocytoses.— By leukocytosis (hyperleukocytosis) is meant a 
transitory increase in the number of leukocytes in the circulating blood 
above the normal maximum figure. For example, 12,000 white cells 
per cmm. would be a leukocytosis. By absolute leukocytosis is meant 
that the actual number of leukocytes per cmm. is greater than normal, 
while by the relative leukocytosis is meant that a certain type is relatively 
increased with respect to the differential count without a marked 
change in the total count. Leukopenia refers to a scarcity in the white 
blood cells such that the total number is below the normal minimum. 
A count of 3500 or 4000 would represent a leukopenia. This occa- 
sionally is referred to as hypoleukocytosis. 

The leukocytoses may be divided according to form of cell which is 
increased. A leukocytosis is ordinarily accompanied by disturbance 
in the proportions of the differential count, so that there is a greater 

1 Xothnagel's Encylopedia of Practical Medicine, Am. ed. 

- States that there are 65 to 75 per cent, polymorpnonuclear forms (neutrophiles and eosinophiles) 
and that 1 to 3 or 5 per cent, of the leukocytes contain eosinophilic granules, and that occasionally 
a larger number is met with in normal blood. 

3 Not given. 

* Included with large mononuclears. 

i Termed by Cabot "large lymphocytes" and including transitional cells. 

6 Termed "endothelial leukocytes." 

' Not stated. 

s Am. Jour. Med. Sc, cxlii, 699-702. 

9 Medical Record, March 25, 1905, lxvii, 453. 

It may well be that Bunting's figures are due to the fact that he was dealing with inhabitants of 
the Great Lakes District, where chronic catarrhal conditions are extremely prevalent. 

The influence of altitude evidently must be considered since Webb and Williams have shown that 
percentage of mononuclears is higher in those living at high altitudes (Tr. Xat. Assn. for Study 
and Prevent, of Tuberculosis, 1909;. 



THE DIFFERENTIAL COUNT 65 

percentage than customary of one or more forms of cell. Accordingly 
we have a polymorphonuclear neutrophilic leukocytosis, an eosino- 
philic leukocytosis (eosinophilia), a lymphocytosis, etc. 

Physiologically, we may have a polymorphonuclear neutrophilic 
leukocytosis in pregnancy, after a cold bath, during digestion, and 
after exeTcise. It is not likely that the count will be increased over 
12,000 or 14,000. It should be determined whether any of the factors 
are present, and if so, due allowances should be made. 

Pathological Leukocytosis.— These are of distinct importance in 
differential diagnosis. The essentials may be presented in the following 
summary. 

Polymorphonuclear eosinophilic leukocytosis is found in infection 
with certain intestinal parasites, e. g., Anchylostoma duodenale, 
Oxyuris vermicularis, Ascaris lumbricoides, and the tenias, in general- 
ized round-worm invasions, such as trichinosis and filariasis, and also 
in echinococcus cyst. It is observed in scarlet fever and is seen 
occasionally in malignant tumors, especially bone-marrow tumors. It 
is expected in postfebrile conditions where there has been a decided 
neutrophilic leukocytosis. Eosinophilia is found after the use of cer- 
tain drugs, such as camphor, and after injections of tuberculin. It 
is found in bronchial asthma, in certain skin diseases such as herpes 
zoster, pemphigus, extensive psoriasis, prurigo, dermatitis herpeti- 
formis, gonococcic invasion of posterior urethra and epididymis, and 
myelogenous leukemia. 

Polymorphonuclear neutrophilic leukocytosis is found in the majority 
of acute infections, such as small-pox, scarlet fever, acute articular 
rheumatism, meningitis (including tuberculous meningitis), cholera, 
erysipelas, diphtheria. In general, a polymorphonuclear leukocytosis 
occurs in infections due to the streptococci, the staphylococci, the 
meningococci, colon bacilli and pneumococci. It is seen after hemor- 
rhage, particularly after post-operative hemorrhage, where there is 
usually a rapid drop to normal, in eclampsia, and after the use of certain 
drugs, such as quinine, salicylates, saline infusion, ether and chloroform 
inhalations, and potassium chlorate in large doses. It is occasionally 
noted with certain malignant growths. In general, neutrophilic 
leukocytosis is seen in abscess and suppurative conditions, unless the 
process is well walled-off . In myelogenous leukemia, the actual number 
of polymorphonuclear neutrophiles is greatly increased, though the 
percentage may be low, on account of the overwhelming number of 
myelocytes. 

Basophilic leukocytosis is seen practically only in myelogenous 
leukemia. 

Lymphocytosis.— An absolute lymphocytosis is seen in lymphatic 
leukemia and often in pertussis, where the count may reach 20,000. 
In infancy and childhood it should be remembered the normal lympho- 
cyte count is relatively high. A relative lymphocytosis is seen in 
pertussis (absolute lymphocytosis also), chronic infectious diseases, 
5 



66 



EXAMINATION OF THE BLOOD 



exophthalmic goiter, pernicious anemia, splenic anemia, cirrhosis of 
the liver, rickets, typhoid fever, measles, uncomplicated tuberculosis, 
influenza, and congenital syphilis. 

Large mononuclear leukocytosis is not frequently seen, though it is 
occasionally noted in severe cases of malaria, the cachexia of carcinoma, 
Hodgkin's disease, and lymphosarcoma. 

VARIATIONS IN DIFFERENTIAL COUNT OF LEUKOCYTES. 



Neutrophilic Leukocytosis. 
(Normally, 60 to 64 per cent.) 



Cholera. 
Small-pox. 
Infection by: 

(a) Streptococci. 

(b) Staphylococci. 

(c) Meningococci. 

(d) Colon bacilli. 

(e) Pneumococci. 
Erysipelas. 
Diphtheria. 

Meningitis, including t.b. meningitis. 

Scarlet fever. 

Tuberculosis with marked sepsis. 

Abscesses, including empyema. 

Majority of purulent wound infections. 

Eclampsia. 

After hemorrhages. 

Following use of certain drugs (inhalations 

of chloroform, ether and use of quinin, 

salicylates, phenacetin, etc. 



A neutrophilic leukopenia ordinarily is 
found when a small mononuclear leu- 
kocytosis is present. 

Small Mononuclear Leukocytosis 
(Lymphocytosis) . 
(Normally, 20 to 25 per cent.) 
Absolute increase: 

Lymphatic leukemia. 
Increase usually only relative: 

Chronic infectious diseases. 

Pernicious anemia. 

Splenic anemia. 

Diseases of the ductless glands. 

Tuberculosis, when uncomplicated. 

Whooping-cough. 

Typhoid fever. 

Influenza. 

Rickets. 

Measles. 

Congenital syphilis. 

Cirrhosis of the liver. 



Eosinophilic Leukocytosis 

(Eosinophilia) . 
(Normally, 2 to 5 per cent.). 

Increase usually relative: 

If there is an actual numerical increase, 
it is slight. 

Certain intestinal parasites (especially 
hook worm; occasionally oxyuris, as* 
caris, and the taenias. 

Trichinosis. 

Gonorrhea, particularly when the poster- 
ior urethra is involved. 

Certain skin diseases, notably prurigo; 
zoster, pemphigus, dermatitis herpeti- 
formis. 

Scarlet fever. 

Bronchial asthma. 

During convalescence from diseases ac- 
companied by neutrophilic leukocyto- 
sis. 

In some cases of tuberculosis, and after 
injection of tuberculin. 

Myelogenous leukemia. 



An eosinophilic leukopenia is usually 
found when there is a neutrophilic leu- 
kocytosis. 

Large Mononuclear Leukocytosis. 



Increase usually only relative: 
Malaria cachexia. 
Cachexia of carcinoma. 
Hodgkin's disease. 
Lymphosarcoma. 



A small mononuclear leukopenia ordin- 
arily is found in the conditions char- 
acterized by a neutrophilic leukocytosis. 



BLOOD IN CHILDHOOD AND INFANCY 67 



THE BLOOD IN CHILDHOOD AND INFANCY. 

We will recapitulate briefly what has been said on this subject 
under different headings with certain additions, in order to present a 
fairly complete summary so that blood findings may be evaluated 
properly. Rigid figures cannot be given, since development is not a 
uniform matter and diverse factors may alter findings even in obviously 
normal children. The red count may be as high as 7,000,000 cells per 
cm. immediately after birth but is usually about 5,700,000. During 
the nursing period the average is about 5,580,000, this count decreasing 
gradually until the proximity of the tenth year, when it reaches 5,100,- 
000, after which time a slight increase may be seen up to 5,500,000. 

The hemoglobin at birth is high, about 135 per cent, according to 
Williamson, dropping rather rapidly to about 75 to 80 per cent, from 
the third to fifth months, and remaining near this figure until the sixth 
year when it tends to gain steadily until the adult standard is attained 
at the tenth year. 

The total white cell count is usually in the neighborhood of 20,000 
to 30,000 or more during the first few days of life. During the nursing 
period, the cells average about 12,000, and a general tendency toward 
diminution is seen. At the sixth year, the count may range from 
5400 to 12,400. The child at the age of fifteen usually presents the 
adult findings in this particular. 

The differential count is quite unlike that encountered in adults. 
It has been shown by Warfield that immediately after birth the poly- 
morphonuclear neutrophiles constitute about 70 to 80 per cent, on the 
first day and it is not until the eleventh day when 40 per cent, or more 
of mononuclears are seen. The findings from the sixth to the twelfth 
month run about 28 to 35 per cent, for the polymorphonuclear neutro- 
philes and 50 to 59 per cent, for the small mononuclears, with a steady 
tendency to assume the adult standard so that the polymorphonuclear 
neutrophiles take the lead at about the third or fourth year, running 
in the neighborhood of 45 per cent, with the small mononuclears 
about 39 per cent. This tendency is maintained so that at or about 
the fifteenth year the adult differential may be expected. Lucas has 
called attention to a change in the blood picture in measles. This 
comes shortly after infection and precedes the earliest signs, i. e., 
Koplik's spots, coryza, or eruption. The leukocyte count falls and 
there is a reversal of the usual relative proportion between neutro- 
philes and mononuclears so that the neutrophiles exceed the mononu- 
clears. 

The general preponderance of lymphocytes in infancy and early 
childhood is due to the greater activity of the lymph adenoid apparatus. 
Furthermore it is to be remembered that the blood in children responds 
to all stimuli much more readily than with adults and that consequently 
high leukocytoses are often seen with comparatively trivial infections. 



68 EXAMINATION OF THE BLOOD 

Moreover, the blood-forming organs may discharge into the circulating 
blood cells (myelocytes, etc.) which are not seen in adults except under 
much graver circumstances. 

SPECIAL METHODS OF EXAMINING BLOOD. 

Microchemical Blood Reactions.— Data of much importance is secured 
by certain microchemical reactions. The demonstration of oxydase 
granules in particular is of importance since it aids us demonstrating 
the origin of the leukocytes. 

Certain leukocytes contain oxydase granules which have the power 
of causing the oxidation of organic substances in the presence of an 
oxidizing reagent. Cells of myeloid origin will give this reaction 
staining blue granules while lymphocytes and lymphoblasts will not. 

Graham's original method may be employed. 

The Oxydase Reaction.— Solutions required: 

s 1 tio \ i 95 per cent- alcono1 9 P arts 

\ Formaldehyde solution (40 per cent, gas), freshly prepared 1 part 

IAlpha-naphthol (Merck's " Recryst "or Merck's "Reagent") 1 gm. 

H 2 2 100 cc. 

40 per cent, alcohol 0.2cc. 

iPyronin 1 gm. 

Anilin 4 cc. 

40 per cent, alcohol 96 cc. 

Solution D (0.5 per cent, aqueous solution Methylene Blue — Grubler's BX.) 

Procedure.— The films should be fixed by covering them with the 
fixative, Solution A. After two minutes, this is washed off with 
water, and the film flooded with Solution B. This is washed off and 
the film is allowed to remain in a dish of running water for fifteen 
minutes. It is then dried and stained for two minutes in Solution C. 
It is again washed in water and Solution D is poured on and allowed 
to remain from thirty to sixty seconds. After washing with water, 
the slide is blotted and mounted in neutral balsam. 

All cells of myeloid origin except the basophilic myelocytes and 
mast-cells show blue granules, while lymphocytes and lymphoblasts 
do not. 

Graham' 8 Second Method.— On account of its more ready availability, 
Graham has suggested the substitution of benzidine for alpha-naphthol, 
dissolving a few crystals (as much as may be taken up on the tip of 
a small knife-blade) of benzidine in 10 cc. of 40 per cent, alcohol, 
adding 0.02 cc. of hydrogen peroxide. The blood smear must be fresh 
and should be fixed with the formaldehyde-alcohol fixative described 
in the previous method as Solution A. This, it is to be noted, should 
be freshly prepared. After it has been allowed to act for a minute or 
two, it is washed off under the tap and replaced with the benzidine 
solution, which is allowed to remain for five or ten minutes. This in 



SPECIAL METHODS OF EXAMINING BLOOD 69 

turn is washed off with tap-water and followed with Loeffler's methy- 
lene blue, which is washed off after thirty seconds. 

With this method the neutrophilic and eosinophilic granules stain 
a warm brown color. The color which appears first is greenish-blue, 
but this remains only momentarily. The neutrophilic granules are 
small and fine and tend to occur in linear aggregations. The eosino- 
philic granule is large and the stained substance appears to be held in 
a mantle or envelope surrounding a relatively unstained central sub- 
stance. The basophilic granules of the mast-cells apparently do not 
react though rare cells, apparently mast-cells, have shown a few 
scattered granules of dense greenish-brown color. The large mono- 
nuclear and transitional cells often have a few indefinite granules of 
brown color. 

Vital Staining. —Demonstration of Skeined or Reticulated Staining.— 
The demonstration of reticulated erythrocytes is the most important 
application of vital staining methods. The stain is readily prepared, 
for it is necessary only to dissolve a few grains of brilliant cresyl blue 
in 1 cc. of normal salt solution, adding a crystal or two of potassium or 
sodium oxalate to prevent rouleaux formation. If the method is 
utilized frequently, a 2 per cent, solution of sodium oxalate in normal 
salt solution may be kept on hand, but the brilliant cresyl blue must 
be added before use. There should be sufficient dye to have the 
solution just translucent in a test-tube 1 cm. in diameter. 

A few drops of blood are taken from the finger or ear and allowed 
to flow into the tube of stain. After gently shaking the mixture is 
put aside for ten to fifteen minutes when a drop of the sediment is 
secured with a capillary pipette, placed on a slide, covered with a 
cover-slip, and examined with the oil-immersion lens. The reticulated 
cells are searched for and their proportion to normal non-reticulated 
cell is ascertained. In any blood there should be visible blue stained 
leukocytes, platelets and hemaconia, but reticulated red cells are seen 
very rarely (less than 1 per cent.) or not at all. They will be recognized 
very readily by the fact that they are somewhat larger than normal 
erythrocytes and show blue stained reticulation. 

On account of the increasing difficulty in obtaining brilliant cresyl 
blue, Buckman and Hallisey have recommended substituting crystal 
violet (penta-methyl-para-rosanilin-hydro-chloride). This stains the 
platelets and the reticular substance of the erythrocytes deep violet. 

Cecil Drinker recommends as the simplest method, placing a drop 
of 1 : 300 solution of cresyl blue upon a slide and allowing it to dry. 
A drop of fresh blood may be placed upon the slide and a cover-slip 
quickly adjusted and rimmed with vaseline. The dried stain is taken 
up quite rapidly; 

Retrculated cells are slightly increased in anemia of any severity 
regardless of type. The increase, however, is quite overshadowed by 
that seen in hemolytic jaundice, when the percentages are frequently 
above 5 per cent, and may reach 15 to 20 per cent. (Pearce) . In per- 



70 EXAMINATION OF THE BLOOD 

nicious anemia the proportion varies from to 20 per cent. (Lee, Minot, 
and Vincent). 

The exact significance of the cells is in doubt, the decision resting 
between nuclear remains of immature cells or products of degeneration 
as part of a disease process. The weight of opinion inclines toward 
regarding reticulation as an evidence of cellular youth. 

Staining of Mitochondria in Lymphocytes.— Mitochondria in the 
lymphocytes may be demonstrated intra vitam by drawing blood into 
a solution of 1:10,000 janus green (diethyl safranin-azodi-methyl- 
anilin). The mitochondria appear as small granules or rod-shaped 
structures in the cytoplasm. Cowdry states that those seen with vital 
staining and those demonstrated in fixed and stained preparations 
have a similar appearance. 

Determination of Coagulation Time.— The determination of clotting 
time is of distinct clinical importance, especially as a pre-operative 
measure in jaundiced individuals or where hemophilia is suspected. 
Of a number of methods which may be employed, it would be imma- 
terial which one were to be chosen if adaptability and cost of equip- 
ment were not items of consideration, provided that the worker be 
thoroughly informed as to the range of normal findings given by the 
method which he employs. 

We give decided preference to the method of Lee and White, on 
account of its simplicity and the inexpensiveness of apparatus required, 
because it can be used at the bedside without a microscope, and because 
the blood is secured without the admixture of tissue juices. 

The apparatus required is a small all-glass syringe of 15 or 20-minim 
(or 1 cc.) capacity with sharp needle to fit, a small glass test-tube 
whose bore is 8 mm., sterile physiological saline solution (0.9 per cent.), 
and the usual articles for preparing for venipuncture. 

The syringe and needle are boiled and after cooling are rinsed by 
drawing in and ejecting the sterile normal salt solution. The test- 
tube is also rinsed out with normal salt solution. Then the skin is 
sterilized over the veins at the bend of the elbow, preferably with 
tincture of iodine followed with alcohol, and blood is obtained with the 
syringe by venipuncture as described elsewhere (page 126). Exactly 
1 cc. of blood should be removed and ejected at once into the prepared 
test-tube, noting the time. The tube is now tipped slightly endwise 
every fifteen seconds until the blood no longer flows but maintains 
its surface contour when inverted. Lee and White have found that 
the temperature is between 65° and 90° F. 

According to this method, the normal coagulation time varies from 
five to eight minutes, with six and a half as an average. Increasing the 
diameter of the tube lengthens the coagulation time. For example, 
Minot and Denny showed that the normal time with a tube 9 mm. in 
diameter is from six to eleven minutes. 

If a specimen of blood be withdrawn by venipuncture into a minia- 
ture Lee and Vincent paraffined transfusion tube for enumeration of 
platelets, erythrocytes, and leukocytes by the Buckman and Hallisey 



SPECIAL METHODS OF EXAMINING BLOOD 71 

method (see page 40), a 1 cc. portion may be withdrawn at once with a 
syringe and placed in a tube of 8 mm. bore to determine the clotting 
time. 

Simple Slide Method.— This method has the advantage of extreme 
simplicity, but is open to the objection that variable and unknown 
amounts of tissue juice are added to the blood, which may materially 
alter the clotting time. An ordinary microscopic slide is cleaned 
thoroughly with soap and water and with alcohol and dried. The ear 
or finger tip is punctured in such manner that the blood will come 
freely with the least possible pressure and a series of eight drops are now 
placed on the slide, noting the time. At the end of one minute a clean, 
sharp, fine sewing-needle is drawn through the first drop, at the end of 
the two minutes through the second, and so on, until a drop is found 
which is picked up by the point of the needle due to a formation of 
fibrin. 

HowelVs Method.— A method similar to Lee and White's has been 
proposed by Howell, according to which 2 to 4 cc. of blood are taken 
and placed in a wide tube (21 mm. in diameter). Coagulation is 
determined by inverting as with Lee and White technic. With this 
quantity and a tube of this size, Howell gives the coagulation time in 
normal individuals as twenty to forty minutes, in hemophiliacs as from 
two and a half to five hours. He states that there is no difficulty after 
venipuncture in hemophiliac patients provided that the vein be not 
wounded. 

Determination of the Bleeding Time.— The coagulation time does not 
always correspond to the bleeding time, probably because of the 
thromboplastin content of the tissues. Duke suggests the following 
method for ascertaining the bleeding time : A small cut is made in the 
lobe of the ear and at half-minute intervals the blood is blotted up 
with filter paper. Each blot represents a half minute's flow of blood, 
and the decrease in the size of the drops is an index to the decrease 
in the hemorrhage. The cut should be such that the diameter of the 
first blot is 1 to 2 cm. The total duration of the hemorrhage is called 
the bleeding time. Normally, it varies from one to three minutes. 

Determination of the Amount of Prothrombin.— Howell has shown that 
the amount of prothrombin is deficient in hemophiliacs. The method 
which he has devised is as follows: The specimens of blood are 
obtained from the superficial veins of the arm by means of a graduated 
syringe of the Luer type. The syringe is of course sterilized before use 
and is partly filled with sterile normal (0.9 per cent, sodium chloride) 
solution. This is expelled so as to leave the needle full in order that 
the barrel may contain no air, since a bubble or two interferes with 
the exact reading. 4 cc. of blood are ejected from the syringe at 
once into a centrifuge tube containing 0.5 cc. of oxalated normal 
salt solution (1 per cent, of sodium oxalate made up in 0.9 per cent, 
sodium chloride solution). The blood is mixed with the oxalate solu- 
tion by inverting the tube, and is then centrifugalized sufficiently to 
obtain perfectly clear plasma. The plasma is pipetted off and 5 drops 



? 2 EXAMINATION OF THE BLOOD 

are placed in each of four tubes. To these tubes are added respectively 
2 6, 4, and 5 drops of 0.5 per cent, calcium chloride solution The 
clotting time of normal plasma is quite uniform, varying between nine 
and twelve minutes in the tubes to which 2 or 3 drops of the calcium 
chloride have been added. In setting up the test, however, it is neces- 
sary to set up a parallel control with normal blood and to subject it 
to the same treatment throughout, since the coagulation time may be 
materially modified by the preliminary centrifugalization. 

The clotting time for oxalated serum to which the optimum amount 
of calcium chloride had been added was markedly prolonged in Howell's 
cases of hemophilia, ranging from ninety to two hundred and fortv 
minutes. There was no variation from normal in purpura hemor- 
rhagica or the other purpuras. The determination of prothrombin 
is of value in differentiating hemophilia from other hemorrhagic con- 
ditions, and is recommended as a preliminary to operation on a patient 
exhibiting the hemophilic tendency. 

Resistance of Red Cells to Hypotonic Salt Solution. -The method is 
based on the fact that though in isotonic salt solution normal red cells 
remain for a period of several hours without damage, normal cells are 
quickly dissolved in distilled water, and hemoglobin is liberated so 
that the fluid is tinged bright red. If intermediate strengths of salt 
solution are arranged, the exact dilution which is required to cause 
hemolysis can be ascertained. This is found to be a variable factor. 

A method which has the advantage of requiring the use of only one 
accurately prepared solution is that proposed by Giffin and Sanford, 
who have modified the technic originally proposed by Ribierre. 

A stock solution is made with great care, dissolving 0.5 gm. sodium 
chloride in 100 cc. of distilled water. A good balance and a volumetric 
flask are prerequisites. A Wassermann rack with two rows of holes 
twelve holes in a row, is filled with tubes. The front row is used for 
the patient's blood, the back for the control. The tubes in the front 
row are marked with a series of numbers, 25, 24, 23, 22, 21, 20, 19, and 
so forth to 14. In each tube is placed the number'of drops of 0.5 per 
cent, sodium chloride solution indicated by the number on the tubes, 
placing likewise the same number of drops in the corresponding rear 
control tube, holding the pipette upright in the same position through- 
out, tor placing the salt in the tubes a capillary pipette is drawn 
from glass tubing or a Mohr pipette may be utilized. This same 
pipette is used for adding distilled water to each tube, though of 
course it should be carefully washed out with distilled water after using 
it tor salt. Enough water should be added to bring the number of 
total drops in each tube up to 25. For example, the tube marked 20 
should contain 20 drops of the saline solution and 5 drops of distilled 
water. 

The patient is bled by puncturing the vein in the manner described 
elsewhere (page 126). An all-glass syringe is to be employed and it 
should be free from moisture. One drop of the patient's blood is 
allowed to flow from the syringe directly into each of the front tubes 



SPECIAL METHODS OF EXAMINING BLOOD 73 

and one drop of the control blood, secured from a normal individual, 
into each of the rear tubes. The tubes should be shaken immediately 
after the addition of the blood. It is highly important that the needles 
used for obtaining the unknown blood and the control blood be of the 
same bore, when the blood is dropped into the tubes of salt solution 
directly from the syringes. 

While the blood may be dropped directly from the syringe into the 
test-tubes, a better method is to secure a definite quantity (about 2 
or 3 cc.) by venipuncture with a graduated syringe and to place it at 
once in a 15 cc. graduated centrifuge tube containing 5 cc. of citrated 
physiological saline solution (2 gm. of sodium citrate to each 100 cc. 
of normal saline). The tube should be corked and inverted gently 
2 or 3 times to wash the cells, when it is placed in a centrifuge and 
revolved until the cells have been packed to the tip. The supernatant 
fluid is pipetted off to leave only cells, which are then diluted with 
normal salt solution to bring the fluid up to the 2 or 3 cc. mark (accord- 
ing to the amount of blood taken originally) . The red cell suspension 
may be added to the tubes with a capillary pipette. By rinsing this 
pipette out with salt solution after use, it may be used for the control 
and any other unknown bloods to be tested at the same time to insure 
uniformity in the size of the drops. The results are recorded after the 
tubes have stood for an hour or two at room temperature. Reading 
from left to right, or from the higher numbered tubes to the lower, 
the tube showing just a slight tinge of color in the supernatant fluid due 
to hemolysis is taken as marking the point of initial hemolysis, while 
the first tube which shows no corpuscular residue on shaking, indicates 
the point of complete hemolysis. The percentage strength of the salt 
solution in the various tubes is easily calculated by multiplying the 
number on the tube by 0.02. 

It is not necessary to have a predetermined normal standard with 
this method on account of the control which accompanies the test. 
Giffin and Sanford, however, have shown that normal blood shows 
beginning hemolysis in 0.42 per cent, to 0.38 per cent, and complete 
hemolysis with 0.36 per cent, to 0.32 per cent. 

The method would seem to be of some value in the diagnosis of 
hemolytic jaundice, since the findings of all workers agree that there is 
an increased fragility, that is, that the cells hemolyze with higher 
strengths of salt solution. With pernicious anemia, with anemias of 
low color index, with splenic and with chronic obstructive jaundice, 
there is an increased resistance. 

Demonstration of Bile Pigments in the Plasma. 1 — Blood is drawn from 
a vein to a test-tube containing potassium oxalate (see page 167). It 
is centrifugalized and the plasma removed with a pipette and placed 
in a small test-tube. Gmelin's test is used, the nitric acid being put 
under the plasma with a fine pipette. The plasma must be free from 
hemolysis. A white coagulum is formed at the junction and in the 

1 Gilbert's method, Compt. rend. Soc. de Biol., 1905. 



74 EXAMINATION OF THE BLOOD 

presence of bile a blue-green ring appears in the midst of the white 
zone. When pigment is small in amount, the color may not appear 
for a half-hour. Blankenhorn found that bile pigment was frequently 
found in pernicious anemia and was the cause of the jaundice. It 
does not appear in the urine because it is fixed in some way to the 
proteins of the plasma. 

THE PATHOLOGY OF THE BLOOD. 

The Anemias.— The general term anemia implies an impairment in 
the quality of the blood. This may be manifested by a reduction in 
the number of erythrocytes or in the amount of hemoglobin or both. 
In considering the anemias we are confronted by a large number of 
clinical conditions between which it is not always possible to make 
sharp distinctions on purely hematological grounds. While many 
cases are so typical that they may be classified readily, a definite pro- 
portion remains in which differentiation is difficult because the blood 
picture is not clearly defined. In this field of diagnosis, as in all fields, 
the final decision (short of that based upon necropsy) rests in the clinic 
and a dogmatic opinion based upon the clinical laboratory findings 
alone often might be quite erroneous. Indeed, it is unfortunate that 
often final diagnosis must be postponed until postmortem pathological 
findings are available. This is illustrated in the discussion of per- 
nicious anemia, where it will be shown that as conclusive as the micro- 
scopic findings in the blood may appear, they may be given in toto 
by the anemia due to dibothriocephalus infestation. This simply 
emphasizes the interdependence between laboratory and clinic, and 
justifies us in the following very brief consideration of the clinical 
features. 

In attempting a classification of the anemias, we perforce adopt 
that which custom has made familiar, for while it is far from satis- 
factory, it fills working requirements fairly well. The anemias are 
divided into the so-called primary or essential anemias and the second- 
ary anemias. In the latter the anemia is a mere symptom of the 
underlying disease which has produced it. The primary anemias are 
of unknown causation and with them the anemia itself predominates 
in the clinical picture. 1 

To simplify the understanding of the response of the blood-forming 
organs to the abnormal conditions which exist in anemias, we have 
found the following comparison helpful. The circulating system of 
the body may be likened to the highways and byways of a great battle- 
front. These roads are filled with volume of traffic of all sorts, similar 
to the blood in the fact that it is quite heterogeneous and made up of 
totally dissimilar constituents. The leukocytes may be likened to 
the soldiers; they are living objects, of different classes, serving different 

x An excellent discussion of the anemias from the modern point of view will be found 
in Minot's article in The Oxford Medicine, the Oxford University Press, New York and 
London, 1920, ii, 589. 



PATHOLOGY OF THE BLOOD 75 

purposes, having different sources. Under abnormal circumstances 
in time of need, the reserves are called upon. For a time these are 
readily furnished, but when they are exhausted, the need for fighting 
material is so great that the homes (deposits of bone-marrow and 
lymphadenoid tissue) are drained for fighting power and immature 
soldiers are sent out, varying in character with the place of their 
origin (myeloblasts and myelocytes, and lymphoblasts) . 

The red cells, whose chief function is the transportation of oxygen 
may be likened to the motor transports, carrying supplies of a certain 
kind, ultimately wearing out, and being sent back to a base (the 
spleen) to be reduced to scrap. In cases of dire emergency, unfinished 
products may be sent out (nucleated red cells) . 

As with all animate objects, the "soldiers" have a certain duration 
of life. Some are lost on the various fronts (mouth, tonsils, bronchi, 
etc.). The blood, however, is not made up of these living cellular 
structures alone. They are carried in the current of traffic (plasma), 
which also carries the food material necessary to feed them and the 
civil population (the body's non-wandering cells), but also the waste 
products which may be sent back through the transport system to be 
gotten rid of at certain appointed stations (kidneys, intestines, lungs, etc.) . 

The Primary Anemias. Chlorosis (Green-Sickness).— Chlorosis, an 
anemia of simple type, is seen in either private or dispensary practice 
rather infrequently nowadays, possibly due to wide use of proprietary 
remedies containing iron. It is regarded as an exaggeration of the 
processes occuring at puberty where normally a change in the blood 
picture is seen as is evidenced by the drop in the erythrocyte count to 
which reference has been made. It is seen then between the ages of 
fifteen and twenty-five years. The diagnosis of chlorosis in mature 
life should not be made without convincing proof nor until other 
causative factors for secondary anemia have been definitely excluded. 
Its occurrence in the male is controversial. There is evidence 
that it has a tendency to afflict several members of a susceptible 
family. The onset is usually quite gradual. The color is not 
the chalky-white of post-hemorrhagic secondary anemia nor is it the 
lemon-yellow tint of pernicious anemia, but there is rather a sallow, 
muddy, pasty, edematous look which is especially marked around the 
eyes. Barker quotes McPhedran's warning about dismissing girls 
with especially red cheeks before examining the conjunctiva and tongue. 
There is no emaciation, indeed such patients are not infrequently 
plump. Among the manifestations which may be seen are digestive 
disturbances; notably perversions of appetite, tachycardia, blowing 
systolic precordial murmurs, nervous disturbances of the so-called 
neurasthenic type with alterations in disposition, headache, edema, 
amenorrhea and skin disorders, such as acne vulgaris. Recurrences 
may occur in later life. 

The erythrocyte count may show little reduction, though counts 
of three to four million are customary. The total number of red cells 
in the body is not diminished, however, since it is generally agreed that 



76 EXAMINATION OF THE BLOOD 

the total amount of blood in the body is actually increased and apparent 
reduction is due to its dilution. The striking point about chlorotic 
blood is the low hemoglobin content. The hemoglobin content of the 
individual cells is reduced. This is made evident by the color index, 
which is of course merely the index of the hemoglobin content of the 
average cell as compared with normal. This may fall as low as 0.5. 
The white cells show no especial change. The platelets are often 
increased in number. In examining stained specimens one is at once 
struck with the red cells which present a greatly increased central pale 
area and sometimes appear as mere hoops or rings of hemoglobin. 
The cells may show slight polychromatophilia and slight anisocytosis. 
Nucleated red cells are not often seen, except in severe cases. 

Diagnosis is comparatively easy when the age of the patient, the 
symptoms, the physical findings, and the blood pictures are viewed 
together. Certain diagnostic pitfalls should be avoided, such as early 
pregnancy, Basedow's disease, gastric ulcer, pulmonary tuberculosis, 
and nephritis. 

Pernicious Anemia. — (Synonyms.— Addison-Biermer type of anemia, 
primary pernicious anemia, progressive pernicious anemia, Addisonian 
anemia, megaloblastic anemia, chronic hemolytic anemia of unknown 
origin.) Fundamentally, the fault is a destruction of erythrocytes 
by an unknown hemolytic factor. The bone-marrow attempts to com- 
pensate for this loss of red cells with more or Jess success for a time, 
but ultimately loses in the struggle. 

The etiology is quite unknown. Anemia of this type progresses to 
a fatal termination, oftentimes with a number of remissions. It occurs 
in middle life or later and in contrast to chlorosis it affects both sexes 
about equally. The onset is usually gradual. The patient's first 
complaint may be of loss of strength, tiring easily, shortness of breath, 
palpitation, or other signs common to any anemia. Certain sensory 
symptoms, however, may predominate such as numbness of the ex- 
tremities, tingling or other parasthesias, and awkwardness in handling 
small objects, and the initial symptoms may be those referable to 
disease of the posterior columns of the spinal cord, such as difficulty 
in locomotion, or retention or incontinence of urine. Dyspepsia or 
even vomiting is a frequent complaint, not to be wondered at when it is 
remembered that achlorhydria is almost constantly present. Intestinal 
symptoms too may be exceedingly difficult to combat, diarrhea being 
not unusual. 

On physical examination we find the remarkable and typical color, 
a distinct lemon-yellow tint, which is sharply in contrast with the pasty 
appearance of the chlorotic. There is little if any wasting. Edema 
is occasionally seen. Hemic murmurs are heard in the precordial area 
and over the great vessels of the neck. Often ulcers of the tongue and 
buccal mucous membranes may be detected. The spleen is at times 
slightly enlarged. Signs of posterior or lateral column degeneration 
of the spinal cord are frequently encountered. Some elevation of tem- 
perature is ordinarily recorded. 



PATHOLOGY OF THE BLOOD 77 

Blood examination shows that the erythrocytes are markedly 
diminished in number, often to 1,500,000 or 2,000,000 even in ambula- 
tory cases. Cabot directs attention to the fact that of 715 American 
cases, 150 did not consult a physician until the red count was below 
1,000,000 cmm. In the terminal stages the count may drop to 150,000. 
The hemoglobin reduction, however, is quite disproportionate, for 
it drops slowly. Consequently the color index is high, practically 
invariably over 1.0, occasionally 1.5 (2.1 in one case). The white cells 
are diminished in number and ordinarily a relative lymphocytosis is 
present. This is understood when we remember that over-activity of 
megaloblastic tissue tends to exclude the leukocyte-forming centers 
from normal activity. The blood platelets, too, are markedly dimin- 
ished in number. This finding may be explained by the deficiency of 




Fig. 22. — Blood in pernicious anemia, showing mitosis in circulating erythroblast. 

the bone-marrow in giant cells. The platelet count increases markedly 
above the normal after splenectomy, occasionally to 900,000 (Lee, 
Minot, and Vincent). The resistance of the red cells is greatly in- 
creased, and an increased proportion of reticulated erythrocytes are 
demonstrable by vital staining methods. 

It is considered (Lee, Mindt and Vincent) that the presence of heavy 
knotted reticulum usually in the center of the cell is of particular 
importance in determining the activity of the bone-marrow. The 
specimens stained by Wright's or similar stains show a remarkable 
picture. The paucity of red cells is discerned, as is irregularity in size, 
shape, and staining (anisocytosis, poikilocytosis, and polychromato- 
philia). The red cells range in size from tiny microcytes to enormous 
macrocytes (2 or 3^ to 12 or 15 fj, in diameter) and vary in depth of 



78 EXAMINATION OF THE BLOOD 

color, when stained with Wright's stain, from the normal coppery- 
pink to a deep blue tinge. Under the staining irregularities should 
be included punctate basophilia (Fig. 22). The deformity of cell 
outline is especially noticeable. Knowing the color-index, one would 
predict the absence of central pale area, which is found to be almost 
universal, the cells being filled with hemoglobin. The occurrence of 
nucleated red cells is expected. These range from normoblasts to 
megaloblasts. Many hesitate to make a final diagnosis until megalo- 
blasts have been demonstrated. To do this, it may be necessary to 
repeat the blood examination on several different occasions. The 
presence of nucleated red cells and of cells with marked polychromato- 
philia is evidence of the abnormal activity of the bone-marrow which, 
while unable to produce normal cells to replace those lost through some 
unknown destructive process, throws immature forms into the circula- 
tion. Methods of vital staining (q. v.) show the presence of reticulated 
red cells whose presence is similarly interpreted. Prothrombin is 
slightly reduced. The reduction is unimportant if active regeneration 
is in process. The urine contains large amounts of urobilin, due to the 
blood destruction. 

At times blood examination may show large numbers of nucleated 
erythrocytes, so-called "normoblastic crisis," evidently representing 
a frantic effort on the part of the bone-marrow to supply new cells. 
After such crises, temporary improvement in the blood findings may 
follow. In certain of the more acute cases a neutrophilic leukocytosis 
may occur. 

Remissions in this form of anemia are seen in a certain proportion 
of cases, during which the general condition is one of apparent health 
and the blood findings may simulate the normal. Of course a remis- 
sion merely represents a pause in the downward course of the disease. 

Aplastic Anemia.— The findings with aplastic anemia, clinical and 
otherwise, are similar to those of primary progressive anemia. Cabot's 
studies of 24 collected cases, shows that it is a disease of young persons, 
that it runs a rapid course with no intermissions, and that death 
usually occurs within a few months from onset of the disease. Sub- 
cutaneous hemorrhage and hemorrhage from the mucous membranes 
are extremely frequent. Fever is a marked symptom. From a 
hematological viewpoint, the chief point of differentiation is the lack 
of any signs whatever of attempts on the part of the bone-marrow to 
bring about regeneration. The blood findings are practically as 
described with the preceding similar anemia, except that nucleated 
cells are rare, reticulated cells are not seen and polychromatophilia, 
stippling, and poikilocytosis may be absent. Leukopenia is the rule, 
and the polymorphonuclear cells are much reduced in number. Plate- 
lets are far below normal, coagulation time may be prolonged only in 
occasional cases, and prothrombin is decreased. (See work of Musser, 
and of Drinker and Hurwirtz, of Cabot, and of Minot.) Post mortem 
shows that in the bone-marrow the erythroblastic tissue has disap- 
peared, leaving bones filled with fat. 



PATHOLOGY OF THE BLOOD 79 

Secondary Anemias.— Anemias Due to Hemorrhage.— After acute 
hemorrhages there is a compensatory transudation of fluids from the 
tissues into the blood which is necessary to maintain the blood volume 
at a proper level. This addition of fluid dilutes the blood and accentu- 
ates the diminution in the number of red cells per cmm., the process 
continues for a short time after the hemorrhage has been arrested. 
When the hemorrhage has not been severe enough to produce death, 
we have prompt evidence in the blood findings of the fact that the bone- 
marrow is striving at regeneration. These may be briefly summarized 
as follows. The red cells^are diminished though not as much so as the 
hemoglobin, since red cells are elaborated more rapidly, so that the 
color index is less than 1.0; the leukocytes are increased, especially the 
neutrophiles (which are of bone-marrow origin). The platelets due to 
the hyperplasia of the bone-marrow are present in greater numbers 
than normal. Occasional polychromatophilic red cells and normo- 
blasts are seen (which indicate the release into the circulation of 
immature forms). 

Chronic Anemias due to repeated hemorrhages or the continued 
destruction of erythrocytes arise from a variety of conditions, notably 
in hemophilia, scorbutus, infection with intestinal parasites, and hemor- 
rhages from mucous membranes (gastric ulcer, epistaxis, hemorrhoids, 
etc.). The blood findings are similar to those in acute post-hemorrhagic 
anemia. The red count and hemoglobin percentage, however, may be 
reduced to lower levels and other changes may be of more severe type. 
There is slight leukocytosis. Some anisocytosis may be seen. With 
the intestinal parasites eosinophilia is found at times. Barker makes 
the point that when the hemorrhage occurs in such situations within 
the body that lost hemoglobin may be reabsorbed from the intestinal 
canal and may be used for the manufacture of new red cells, the color 
index is not so low and punctate basophilia of the reds is more pro- 
nounced. 

Anemias Occurring in Malignancy and the Puerperium.— Anemias due 
to these causes are ordinarily of the type seen in chronic hemorrhages, 
but occasional exceptional instances of anemia make the recognition of 
these possible causative factors all the more important. With cancer 
we have to do with two factors, the loss of blood by hemorrhage and the 
destruction of blood by hemolytic substances. In cases where the 
new growth is situated in the digestive tract, an added factor may be 
starvation due to interference with the digestive process. The anemia 
in sarcoma cases is frequently extremely severe, and that due to the 
invasion of the bone-marrow by metastases of malignant tissue are 
particularly profound. There are instances where the color index is 
not markedly lowered and where the blood findings consequently verge 
upon those seen in pernicious anemia. 

Anemias Resulting from Infections.— Anemia is seen with practically 
all acute febrile conditions. It is likely to be especially marked with 
acute articular rheumatism, typhoid fever, malaria, acute septic 



80 EXAMINATION OF THE BLOOD 

conditions, chronic suppurations, syphilis and tuberculosis. The 
factors may be the actual destruction of erythrocytes by the infecting 
organism (e. g., streptococcus hemolyticus, malarial plasmodium, etc.), 
to the interference with nutrition due to limitation of diet or to 
actual loss of blood by hemorrhage. In these cases the anemia is 
usually of the so-called "secondary" t\Tpe just described. 

Anemia is seen in certain cases infected by the dibothriocephalus 
latus. The anemia may be of the chlorotic or secondary type with 
low color index or it may be indistinguishable from pernicious anemia, 
showing high color index, poikilocytosis, polychromatophilia, punctate 
basophilia, and nucleated red cells, including even megaloblasts. 
The liberation in the intestinal tract by decomposed segments of the 
worm of a toxic substance and its absorption by the host is held by 
some (Tallquist and Faust) to produce the blood destruction. 

Anemia Due to Intoxications.— Anemia may be produced by certain 
inorganic poisons, such as mercury, lead and arsenic, by autogenous 
poisons generated in chronic affections, such as nephritis and jaundice, 
and by benzol. 

Of especial interest is the anemia produced by benzol because of the 
trial which it was given as a therapeutic agent in myeloid leukemia 
and because of the poisoning resulting from its use in various industries, 
especially where rubber is handled. The problem of its effect upon the 
hematopoietic organs has been exhaustively studied by Selling. The 
clinical features presented are a purpura hemorrhagica with skin 
lesions, retinal hemorrhages and bleeding from the mucous membranes. 
The symptoms of the poisoned cases are those of a marked toxemia, 
with high fever and rapid pulse and before death in fatal cases delirium 
( Harrington) . The anemia is of the aplastic type, the red cells reach- 
ing 640,000 in one case .(Selling), and 1,150,000 in another. The 
red cells show only slight changes, chiefly anisocytosis, without poikilo- 
cytosis. There is little evidence of regeneration as shown by the 
absence of nucleated forms. Blood platelets are scanty. There is a 
decided leukopenia, counts as low as 140 before death being reported 
(Selling) . The polymorphonuclear percentage is greatly decreased and 
the mononuclear cells relatively increased. 

The anemia produced by lead poisoning is also of practical interest 
on account of the frequency of plumbism in the trades, notably among 
those who handle lead paint, in battery-makers, metal-polishers, and 
compositors. The red corpuscles fall to three and a half million or less, 
and the hemoglobin falls more or less in proportion. There may be a 
slight leukocytosis or a slight leukopenia. The striking feature is the 
presence of basophilic stippling of the red cells, so-called punctate 
basophilia. 'While this has been regarded by some as pathognomonic 
of lead poisoning, it is not, since it is found in other conditions, notably 
pernicious anemia or the more severe secondary anemias. Some 
writers have maintained that the presence of 100 basophilic red cells 
per million corpuscles should lead the factory physician to watch the 



PATHOLOGY OF THE BLOOD 81 

worker's health closely, and the presence of 300 per million to cause his 
suspension from work. There are many definite cases of plumbism, 
however, which do not show basophilic granulation of the red cells, 
and its absence in a doubtful case is not to be considered as excluding 
lead poisoning from diagnostic consideration. Granulation may appear 
promptly after the ingestion of lead. White and Pepper showed it 
might be found in the peripheral blood twenty-five hours after the 
ingestion of 7.5 grains of lead acetate. 

Anemias Due to Poor Hygienic Surroundings (Anemia of the poor).— 
This type is frequently encountered in hospital and dispensary practice. 
Overcrowding and the consequent lack of fresh air, the deficiency of 
food, particularly of varieties rich in iron, and overwork are the factors 
which lead to inanition and subsequently deficient production of blood, 
producing a mild or moderate type of secondary anemia. 

Leukanemia.— This term is rapidly passing into disuse since it is 
felt that most cases whose blood pictures combine the features of 
leukemia and pernicious anemia are either leukemia with terminal 
anemia or pernicious anemia with lymphoid marrow. 

Other Anemias Showing Splenomegaly.— In addition to the anemias 
already described, there are certain syndromes presenting, among other 
features, splenic enlargement and secondary anemia. Brief con- 
sideration of them is in order, simply because of the anemia. The 
literature on this subject has been carefully studied by Pearce and 
co-workers. The table presented on page 83 from their work gives 
a resume of the conditions in which splenomegaly and anemia are 
coexistent. 

Gaucher 's Disease.— The onset is in infancy or childhood and is 
insidious. The course is chronic, usually about twenty years. Later 
there is distinct anemia, subcutaneous and submucous hemorrhages, 
and progressive enlargement of the spleen. There is nothing charac- 
teristic about the anemia. It is of the chlorotic type, with definite 
leukopenia. 

Banti's Disease.— It usually occurs in young and previously healthy 
adults. The course is chronic. First there is a period marked by in- 
creasing pallor, weakness, digestive disturbances and abdominal pain. 
It may be noted now that the spleen is enlarged, smooth, and hard. 
A tendency to hemorrhages is seen. The anemia is moderate, of the 
chlorotic type, with no distinguishing characteristics. The resistance 
of the red cells is normal. Leukopenia is the rule. 

Later, there is a slight increase in the size of the liver; the urine is 
small in amount, highly colored, and contains much urobilin. Diar- 
rhea and dypsepsia become bothersome. Finally symptoms of liver 
cirrhosis predominate the picture, there is more or less ascites, slight 
jaundice, increasing anemia, and emaciation. Death is by hemorrhage 
or intercurrent infection. 

Anemia Pseudoleukemia Infantum (v. Jaksch's Disease).— This 
condition is occasionally encountered in infancy and early childhood. 



S2 EXAMINATION OF THE BLOOD 

Exact classification is difficult, since the same term is used to include 
many different pictures and probably should not be employed at all. 
In fact, like the term leukanemia, it is rapidly disappearing from 
current literature. Attention has been directed to the prompt response 
of infantile bone-marrow to any stimulus. One would therefore expect 
to find in a variety of conditions nucleated red cells and myelocytes 
in the circulation under much lesser provocation than is required to 
bring them out in adult life. 

In cases which have been described as V. Jaksch's disease, the spleen 
is large, smooth, and hard. The red cells are much reduced in number, 
possibly as low as one million. The hemoglobin is not reduced pro- 
portionately, so the color index is over 1.0. There is marked aniso- 
cytosis, polychromatophilia, and punctate basophilia, and many 
nucleated red cells both of normoblastic and megaloblastic type. 
The leukocytes are usually increased in number, from 20,000 to 100,000 
per cmm., the neutrophilic leukocytes predominating. Myelocytes 
may be seen. The blood findings therefore are at first quite confusing. 
It is stated that the condition may develop into anemia, into myeloid 
leukemia, or terminate ultimately in recovery. Doubtless this simply 
means that the condition was originally a more or less severe anemia 
or a leukemia, that the blood picture was confused for the reasons 
already stated, and that time was required for the typical picture to 
appear. In interpreting the results of blood examination in infants 
and children, it is always well to remember the readiness with which 
the bone-marrow responds to stimuli and to make suitable allowances 
in evaluating seemingly grave findings. 

Hemolytic Jaundice.— 1. The acquired type of hemolytic jaundice 
(Hayem-Widal) is characterized by chronic jaundice with the presence 
of bile pigment in the blood serum though not in the urine, and by 
splenic enlargement. There are no signs of obstructive jaundice, such as 
clay-colored stools and itching. The spleen is large and hard, the liver 
possibly slightly enlarged. The anemia may be sufficiently grave to 
dominate the clinical picture. The blood picture is often similar to 
that seen in pernicious anemia, with high color index, anisocytosis, and 
poikilocytosis. The leukocytes, however, may be increased in number. 
(For discussions of hemolytic jaundice see the reports of Thayer and 
of Tileston.) Counts below 1,000,000 are recorded in cases (Pearce). 
Increased numbers of microcytes and of reticulated red cells have been 
reported, pointing to the attempt at regeneration. The red cells show 
decreased resistance to hypotonic salt solution. There are periods of 
exacerbation, termed by Widal "crises of deglobulization" in which the 
jaundiced becomes more intense and bile appears in the urine. 

The exact pathogenesis is the subject of debate, some holding that 
the fundamental fault is in the blood cells (a dystrophy of the blood 
cells), others that there the spleen has an exaggerated hemolytic 
activity. Marked improvement follows splenectomy according to 
Pearce, Krumbhaar, and Frazier. 









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84 EXAMINATION OF THE BLOOD 

2. The familial type of hemolytic jaundice (Chauffard-Minkowski) is 
quite similar to that just described, but there is a history of other cases 
in the family and of symptoms dating from early life. The spleen is 
enlarged but certain clinical differences are seen. The acquired form 
usually starts with an acute illness and the patient is anemic rather 
than jaundiced. In the familial type, jaundice may be noted but it 
may be lightly regarded by the patient and dismissed by him as a 
family characteristic which does not interfere with his health. 

The blood picture is not usually as severe as that of the acquired 
type, the average count of 104 cases being 3,340,000, ranging from 
1, 800,000 to 5,700,000. When severe, it may be indistinguished from 
pernicious anemia. 

The Leukemias.— In leukemia there is extensive permanent hyper- 
activity of the tissue producing leukocytes with escape of an excess of 
cells into the circulation. The transitory leukocytoses accompanying 
certain infectious diseases are not included in this heading. Either 
the lymphatic or the myeloid tissue may be involved, and the condition 
is accordingly referred to as lymphatic (lymphoblastic) or myelogenous 
(myeloid, myelocytic, splenomyelogenous) leukemia, respectively. In 
either case the condition may be acute or chronic, the acute case^ 
being relatively rare. 1 

Chronic Myelogenous Leukemia.— The onset is usually insidious, the 
patient noticing first the abdominal tumor, or abdominal enlargement 
due to the increased size of the spleen, though gastric-intestinal 
symptoms, shortness of breath, or some symptoms due to anemia may 
be prominent early complaints. The blood is fairly characteristic. 
Its whiteness may be discerned grossly, and the preparation of satis- 
factory films is often difficult because of its creamy consistence which 
makes the drop spread slowly. The leukocyte count is usually high, 
occasionally reaching 1,000,000 per cmm. (Osier). Frequently an 
anemia of the secondary type is seen, which may range in intensity 
from slight to extremely severe. This may be accounted for by the 
hyperplasia of the myeloid marrow tissue, which crowds out erythro- 
genic tissue. The stained specimens give a striking picture, the leuko- 
cytes occasionally equalling or even exceeding the red cells in number. 
All types of leukocytes show an absolute increase. The predominant 
feature is the presence of myelocytes (see preceding description), or of 
myeloblasts, (immature non-granular myelocytes) which may com- 
prise 30 to 50 per cent, of the total number of leukocytes. Nucleated 
red cells are seen; usually they are normoblasts, though megaloblasts 
are not infrequently seen. The blood picture is subject to considerable 
variation, since most cases have a course marked by many ups and 
downs. I Hiring remissions, especially after a>ray or radium treatment, 
the blood findings may be practically normal, in regard to both the 
leukocyte count and the differential formula. During the course of 

1 The recent article by Ordway and Thomas should be consulted by the student. 
It will be found The Oxford Medicine, the Oxford University Press, New York and 
London, 1920, ii, 681. 



CLINICAL SIGNIFICANCE OF BLOOD EXAMINATIONS 85 

acute infections, the differential count may show a preponderance of 
neutrophilic polynuclears. 

Chronic Lymphatic Leukemia.— Ordinarily the first symptom noted is 
an enlargement of the lymphatic glands, usually beginning in the 
cervical region. There is often a history of trauma. Some splenic 
enlargement may be seen and an anemia of the secondary type develops. 
The lymphatic enlargement may be present for some time before a 
diagnosis is made for the truth may be revealed only by a routine blood 
examination. 

The leukocyte count averages from 100,000 to 500,000 per cmm., 
ranging from less than 10,000 to over 1,000,000. Cabot calls attention 
to the large proportion of low or moderately low counts in his series, 
comparable to the leukocytosis of infectious disease, and to the likeli- 
hood of such counts being overlooked in a hasty examination. Stained 
films show the predominance of lymphocytes, which make up 90 per 
cent, or more of the total number of leukocytes. Ordinarily these 
correspond to the small mononuclears or small lymphocytes of the 
normal blood, but occasionally there may be lymphoblasts and many 
lymphocytes, the latter, identical in all respects with the small 
lymphocytes except for their greater size. 

As with myeloid leukemia, the course of the disease may show many 
remissions during which the blood picture varies, though there is 
ultimate progression to a fatal outcome. The oxydase reaction is of 
assistance in doubtful cases in demonstrating that the mononuclear 
cells have no oxydase granules and therefore, are not myeloid cells. 
Acute Myelogenous Leukemia.— This condition is relatively rare. It is 
marked by a fulminating, febrile course, advancing rapidly to fatal 
termination. The blood picture may be similar to that of chronic cases. 
Myeloblasts may predominate, however, and the granular myelocytes 
may be absent or few in number, so that confusion may arise, and 
recourse to special staining methods (see oxydase reaction) may be 
necessary to determine the origin of these non-granular leukocytes 
and so to establish the fact that one is dealing with acute myeloid 
leukemia rather than with acute lymphoid leukemia. 

Acute Lymphatic Leukemia.— The onset is usually sudden, marked 
by throat symptoms such as difficulty in swallowing, swollen gums, 
high temperature, hemorrhagic signs, and later by swelling of the 
glands. The white count increases rapidly up to 500,000 per cmm. 
(Barker). The cells are often chiefly of the large lymphatic type. 
There is an early fatal termination. 

THE CLINICAL SIGNIFICANCE OF THE FINDINGS OF BLOOD 
EXAMINATIONS. 

The interpretation of the findings is a matter of judgment, which 
must have as its basis clinical experience. Indeed diagnosis of the 
anemias often rests as much upon clinical grounds as upon considera- 
tion of the laboratory findings. These have been considered rather 
fully under the appropriate headings. 



86 EXAMINATION OF THE BLOOD 

As may be seen from the discussions in the preceding sections, many 
factors influence both the actual number of leukocytes and also their 
relative proportions. Many of the findings taken by themselves are 
of little moment, since in general, any bit of information, clinical or 
laboratory, is valuable in inverse proportion to its frequency. For 
example, polymorphonuclear neutrophilic leukocytosis is found in a 
variety of conditions so that a correspondingly large list of possible 
diagnoses may be considered when it is found. Eosinophilia, on the 
other hand, is seen less often. Its occurrence leads often to the correct 
conclusion with promptness. In practically no instance, however, 
except with parasitic blood diseases, do we have findings which are 
absolute. Almost invariably, they must be taken as part of the 
clinical evidence and weighed as such. 

Leukopenia in a febrile condition suggests the possibility of typhoid 
fever, malaria, or tuberculosis infection. In the early stages of the 
exanthemata, a differential count may be of service; for example, in 
differentiating between measles and scarlet fever. Typically, measles 
shows a lymphocytosis and no eosinophilia; scarlet fever manifests a 
polymorphonuclear leukocytosis and often an eosinophilia. 

When typhoid fever is suspected, leukopenia is of significance in 
differentiating from other acute infections except tuberculosis or 
malaria. During the first ten days blood cultures (q. v.) may serve to 
establish the diagnosis while later, agglutination tests (q. v.) are of 
more value. During the course of a typhoid infection it is worth while 
to make a count of the white blood cells at stated intervals, say twice 
weekly, to determine the level which the count maintains, since 
symptoms of perforation or hemorrhage may make a count desirable 
and interpretation would be more reasonable when based on a knowl- 
edge of preexisting conditions. 

In septicemia, there is usually a leukocytosis with increase in the 
number of polymorphonuclear cells. Blood cultures (q. v.) frequently 
reveal the infecting organism. 

Cases of pulmonary tuberculosis with few exceptions show a chloro- 
anemia, characterized by a normal or slightly subnormal number of 
erythrocytes with a markedly decreased percentage of hemoglobin. 
With a moderate or slight degree of involvement there is no increase 
in the number of leukocytes. The more advanced cases show a 
leukocytosis of only slight degree, unless there are complications. In 
the earlier stages and with a small degree of involvement, the per- 
centage of lymphocytes is higher and that of polymorphonuclears 
smaller while the more advanced and unfavorable cases show a lower 
percentage of polymorphonuclears, according to Craig and M. Solis- 
Cohen and Strickler. This checks well with the fact that the lympho- 
cytes play a definitely protective role in tuberculosis. 

In regard to the interpretation of a leukocytosis, it should be remem- 
bered that leukocytosis is largely dependent upon resistance of the 
body against infection, while the increase in the proportion of poly- 



CLINICAL SIGNIFICANCE OF BLOOD EXAMINATIONS 87 

morphonuclear cells in the differential count is taken as an index to the 
severity of the infection. (Sondern.) 

Generally, therefore, a sharp leukocytosis is a hopeful sign in those 
conditions in which it is ordinarily expected; conversely, its absence in 
such instances is of poor prognostic import. For example, a pneu- 
monia patient with a count of 20,000 or 25,000 does not cause the 
attending physician the anxiety that one would with a count of 8,000, 
others conditions being equal. 

Sondern has drawn the following conclusions from a study of counts 
in pneumonia cases: 

1. Slightly increased polynuclear percentage indicates slight toxic 
infection, regardless of the leukocyte count. 

2. Greatly increased polynuclear percentage indicates severe toxic 
infection, irrespective of the leukocyte count. 

3. Slight leukocytosis with slight polynuclear increase indicates fair 
resistance and slight toxic infection. 

4. Pronounced leukocytosis with slight polynuclear increase indicates 
good resistance and a slight toxic infection. 

5. Slight leukocytosis with pronounced polynuclear increase indi- 
cates poor resistance and a severe toxic infection. 

6. Pronounced leukocytosis with pronounced polynuclear increase 
indicates good resistance and a severe toxic infection. 

7. Absence of leukocytosis with pronounced polynuclear increase 
indicates no resistance and severe toxic infection, and a falling leuko- 
cytosis with a rising polynuclear percentage indicates diminishing 
resistance and increasing toxic infection. 

8. Falling leukocytosis with a falling polynuclear percentage indi- 
cates diminishing toxic infection or recovery. 

It has been definitely shown that the mortality in pneumonia cases 
with a leukocyte count below 10,000 is 55 to 60 per cent, and that 
with a rise in the leukocyte count from 10,000 to 30,000 there is a 
progressive decrease in the mortality (Chatard, Avery, Chickering, 
Cole, and Dochez). When consolidation of the lobe or lobes is taking 
place, or during the period when a spread of infection to a new area of 
lung is in progress, there may be a well marked decrease in the number 
of leukocytes, even to a point below 10,000. A steady rise in the 
leukocyte count is usually of good import. A continuance of a high 
count even after crisis, especially if associated with a high percentage of 
polymorphonuclear cells, suggests some complication with pus forma- 
tion. Continued leukocytosis with diminishing polymorphonuclear 
count, correspondingly increased lymphocyte count, and the presence 
of a few eosinophiles, suggests delayed resolution, or serum sickness, 
if the patient has been treated with specific serum. (Avery, Chick- 
ering, Cole, and Dochez.) ■ 

Accepting Sondern's hypotheses regarding the significance of in- 
creased total leukocyte count and increased polymorphonuclear per- 
centage, Gibson has advanced a theory which is the basis for a curve, 



88 EXAMINATION OF THE BLOOD 

arranged to show the disproportion between total leukocyte increase 
and increase of polymorphonuclear -percentage. He assumes as 
standard a leukocyte count of 10,000 and a percentage of polymor- 
phonuclear cells of 75. Paper ruled in squares is used for plotting and 
the horizontal base line is taken as representing a count of 10,000 
white cells and likewise an increase of 1 per cent, in the polymor- 
phonuclear cell count. The white count is plotted on the left hand 
side of the sheet and the percentage of polymorphonuclear cells on the 
right hand side. A line is drawn from point to point. When the 
proportion is normal, the line connecting the two points is assumed to 
be approximately horizontal. A rising line is interpreted as indicating 
a rather more severe lesion and less resistance, and if the interval 
between the two points is considerable, for example, ten squares or 
more, this fact is taken as indicating a severe lesion. A line horizontal 
or approximately so, indicates that the lesion, even if severe, is well 
borne. 

Certain writers claim to have found the Gibson curve useful, but 
have objected to the complexity of lines which the chartering of con- 
tinued observations would entail. Walker has suggested the use of 
an index, since the "resistance line" is simply the visual expression of 
the disproportion between the total leukocyte count and the poly- 
morphonuclear percentage. He assumes that the same arbitrary 
standard for the white normal that Gibson does and takes 70 normal 
percentage for polynuclear cells. The formula is: 

(T-10)-(P-70)=I.R. 

T is the digits in the thousandths place of a given leukocyte count; 
10 is the digits in the thousandths place of the high normal leukocyte 
count; P is the percentage of polymorphonuclear cells in the differen- 
tial count of the same patient at the same time; 70 is the high normal 
polymorphonuclear percentage; and I.R. represents the "index of 
resistance." As an example, if the total leukocyte count were 15,000 
and the polymorphonuclear percentage 80, substitution in the formula 
would give (15-10) - (80-70) = -5 =I.R. This is a negative index, 
indicating according to the theory a severe infection which is com- 
paratively greater than the resistance of the body to it and corresponds 
to a rising line on the Gibson chart. If, however, the total leukocyte 
count is 25,000 and the percentage of polymorphonuclear cells 75, 
substitution in the formula would make the index of resistance +10, a 
positive index, corresponding to a falling resistance line, and pointing 
to a good reaction to a comparatively mild infection. The use of the 
index of resistance in this manner makes transference of the results 
to graphic charts a simple matter. Enough work with the Gibson 
curve has not been reported to make it possible to determine finally 
its clinical value. 

In surgical conditions, Sondern has defined three distinct pictures: 
First, a relative percentage of polymorphonuclear cells below 70 per 



CLINICAL SIGNIFICANCE OF BLOOD EXAMINATIONS 89 

cent. With an inflammatory leukocytosis of any degree, he feels 
that this excludes the presence of pus or gangrene and points to good 
body resistance against infection. Second, an increased relative pro- 
portion of polymorphonuclear cells with little or no inflammatory 
leukocytosis is an absolute indication of the inflammatory process. 
The percentage is a direct guide to the severity of the infection. In 
adults, a purulent exudate or gangrene is uncommon with a poly- 
morphonuclear percentage below 80 per cent, and a percentage over 85 
per cent, is rare without purulent exudate or gangrene. In children, 
the percentage of polymorphonuclears is less than with adults and pus 
is sometimes seen with a percentage as low as 73. Third, an increased 
percentage of polymorphonuclear cells and a decided leukocytosis. 
Most of the cases of inflammatory lesions fall into this class. Here the 
percentage of polymorphonuclear cells is held to be a guide to the 
status of the inflammatory lesion. In following such cases, the Gibson 
curve (q. v.) might serve to present the changes from time to time in a 
graphic way. 

The Arneth Formula.— Arneth has described very fully the possible 
variations in the nuclear arrangement of the polymorphonuclear 
neutrophilic leukocytes. He has divided them into five groups accord- 
ing to the number of nuclear lobes, those in Group I have one nuclear 
lobe, those in Group II have two nuclear lobes, etc. In the first group 
are placed the mononuclear forms, subdividing this into (a) those with 
a single unindented nucleus, corresponding to Ehrlich's myelocytes, 
(b) those with but a slight indentation of the nucleus (metamyelo- 
cytes), (c) those which the indentation has extended further than in 
the preceding group but is not sufficient to separate it into two really 
distinct portions. Cells of the first types, a and b, are not seen in 
healthy subjects while those of the type c normally compose about 5 
per cent, of the neutrophiles. 

The second group, comprising cells with two distinct nuclear seg- 
ments, constitutes in normal cases about 35 per cent, of the total 
neutrophiles. Cells of the third group, characterized by three nuclear 
portions, are usually present to the extent of about 41 per cent, of the 
total neutrophiles, while cells, those of the fourth group, having four 
nuclear fragments, ordinarily form 17 per cent, of the neutrophiles. 
Cells of the fifth group have five or more nuclear subdivisions and 
normally form about 2 per cent, of the total. It has been assumed that 
cells pass through various stages in the process of development, that 
the cells of the first group are the youngest in the point of development 
and those of group V the most mature, the other groups representing 
intermediate stages. Arneth's clinical deductions have not received 
universal support. 

In recording the results of the count the following scheme is usually 
employed: 

Group No I. II. III. IV. V. 

Percentages 5 35 41 17 2 



90 EXAMINATION OF THE BLOOD 

To make graphic representation of the results possible, Arneth took 
the sum of the cells in the first two groups as the index. In this 
country the index proposed by Bushnell and Treuholtz is more com- 
monly used. This takes as the index the sum of the cells in the first 
and second groups and one-half of those in the third group. There is 
some disagreement as to the exact index for normal individuals, this 
fact pointing to the influence of personal equation in classifying the 
nuclear divisions. The normals obtained by various workers are 
Arneth, 60.5 (when his figures are translated to terms of Bushnell and 
Treuholtz 's standard); Briggs, 57.73; Bushnell and Treuholtz, 67; 
Miller and Reed, 48, with a normal range from 45 to 55; Minor and 
Ringer, 48.5. An increase in the index is spoken of as a "shift to the 
left," meaning of course, an increase in the number of cells in the first, 
second and third groups. By many workers, this "shift to the left" 
is interpreted as indicative of low resistance in tuberculosis. They 
hold that there is a return of the cells in the groups to the right is 
found with improvement. This point, however, is not universally 
accepted, and is opposed by Cohen and Strickler. 

Dbhle Inclusion Granules.— Dohle has described the occurrence in the 
polymorphonuclear neutrophiles of small coccus-like or rod-shaped 
bodies, usually situated at the periphery of the cell. While they may 
be demonstrated with Wright's or Giemsa's stain, it is stated by some 
workers that the use of pyronin-methyl-green is preferable. With 
this stain, the Dohle bodies stain bright red and the nuclei of the cells 
purple. While the bodies appear to be present in scarlet fever with 
considerable constancy, they are also found in other conditions. The 
absence in suspicious cases rather tends to exclude scarlet fever, but 
their presence can not be taken as conclusive confirmatory evidence. 

For demonstration of the bodies, Pappenheim's methyl-green and 
pyronin or Granger and Pole's modification of Manson's stain may be 
employed. The former is prepared by mixing two parts of a saturated 
aqueous solution of pyronin with four parts of a saturated aqueous 
solution of methyl-green. With this stain the inclusion bodies stain 
bright red and the nuclei a greenish-blue or purple. Granger and 
Pole's modification of Manson's stain is made as follows: Methylene 
blue, 1.5 gm.; absolute alcohol, 10 cc; 5 per cent, solution of phenol 
in water, 100 cc. In either case, fixation should be accomplished by 
flooding the slide or cover-slip with absolute methyl alcohol, which is 
allowed to remain for two minutes. The nuclei and the inclusions stain 
a deep blue and the cytoplasm a pale blue with this stain. While the 
bodies appear to be present with considerable frequency in scarlet 
fever, especially up to the fourth day and even to the tenth day, they 
are also found in diphtheria, pneumonia, tuberculosis, sepsis, and 
severe streptococcal angina. They are not found in serum rashes or 
in toxic rashes due to other causes than serum, nor have they been 
found in German measles. Definite cases of measles from the pre- 
emptive stage through the first week do not show a picture similar to 



EXPLANATION OF PLATE IV. 

(Park and Williams' Pathogenic Microorganisms) . 

Partly schematic. Drawn and rearranged by Williams, partly from Muir and Ritchie, 
partly from Kolle and Hetsch and partly original. Giemsa's stain. 

The asexual forms show cycle of the organism in the red blood cells of the human host. 
They show schematically the time of fever and the day of segmentation. 

Tertian type. 

Fig. 1.— Segmented organism. 

Fig. 2.— Young ring form in cell and a young form on surface. 

Fig. 3.— Growing schizont; irregular form due to great motility; beginning pigment 
formation; red blood cell becoming paler. 

Fig. 4. — Larger schizont with dividing nucleus. Red blood cells pale and stippled. 

Fig. 5.— Nucleus divided into four clumps. 

Fig. 6.— Further division of chromatin and formation of irregular rosette. Pigment 
finely granular in center. 

Fig. 7.— Segmentation. Note eighteen merozoites (usually sixteen). 

Quartan type. Shows following differences from tertian. Slightly larger, fewer seg- 
ments (usually eight), and more regular. Pigment coarse. Red blood cells unaltered. 
Segmentation every seventy-two hours. 

Estivo-aulumnal type. Shows following differential points: Merozoites smaller and 
more numerous (thirty-two?); organism less motile with less pigment. Red blood cells 
smaller and greenish color (in fresh cells). 

Sexual Forms. Show cycle of development in mosquito. 

Fig. 1 (A to E).— Male (tf) and female (?) forms of tertian type formed in human 
blood; F, flagellation of male type in stomach of mosquito; G, H, changes in female type 
and fertilization in stomach of mosquito. 

Fig. 2. — Development of sporocyst within mosquito. Liberation of sporozoites, which 
find their way to the salivary gland. 

Fig. 3. — Sexual forms of estivo-autumnal type found in human blood, showing devel- 
opment of sickle-shaped bodies. 



PLATE IV 



ASEXUAL FORMS OF MALARIAL PARASITES 
(SCHIZOGONY) 
TERTIAN TYPE 



1 m M p. 






2 DAYS 

QUARTAN TYPE 



> a» % (*) g» §$*$ 



2 
1 



2 DAYS 

/EST I VO -AUTUMNAL TYPE 



SflR? 2 



I DAY 

7 



° ■> c 

$ * ? 







■4- 


5 


6 


* » • " • 








2 DAYS 






3 DAYS 








SEXUAL 


FORMS 






, 




2 










* 






i "'">. 


*• ' ; , J 






--/•■•. 

' ♦ .:■»■ 



. . 

(i-m 3r« «;»v\ 



F ik -* tee. 



■•;« 






* •% ! «* P ^#, 



C D + E F Q H 



DISEASES IN WHICH THE BLOOD CONTAINS PARASITES 9l 

scarlet fever, though tiny granules are found in the polymorphonuclear 
quite unlike the larger granules seen in scarlet fever. From the work 
which has been done by Kolmer and Nicoll and Hill it would appear 
that the absence of the bodies in suspicious cases tends to exclude 
scarlet fever but that their occurrence cannot be taken as conclusive 
evidence of its presence. 



DISEASES IN WHICH THE BLOOD CONTAINS PARASITES. 

Malaria.— In malarial infection, man serves as the intermediary host 
for an infection by one of three forms of plasmodium, the definitive 
host for the infecting organism being the female Anopheles mosqi ito. 
The plasmodium may be of three species, P. vivax, P. malarise, and P. 
falciparum giving rise respectively to the so-called benign tertian, 
quartan, and estivo-autumnal clinical varieties of the malaria. In 
man, only the asexual cycle of development takes place, and the sexual 
forms which are seen in the circulating blood are simply the gametes 
which develop from the asexual parasites, and whose development 
goes no further in the human host. 

Asexual development proceeds by geometrical progression after the 
entrance into the circulation of the first sporozoite, but ordinarily 
there are not enough parasites to produce a paroxysm until about two 
weeks after infection. The paroxysm takes place at the time when the 
schizont (presegmenting forms) rupture and the paroxysm is thought 
to be due to a toxin set free by the rupture of the schizont. 

In examining blood for malaria, fresh unstained specimens may be 
employed. This method has the advantage of permitting observation 
of ameboid movement; but it has the disadvantages that the examina- 
tion must be carried out practically at the bedside and that recognition 
of the unstained parasites is not especially easy unless one has had 
much practice with this particular method. Therefore, the use of a 
suitable stain is recommended. Wright's stain is most useful as it 
stains the chromatin and is well adapted to show all the details of the 
parasite. 

When the clinical signs give rise to the suspicion of malaria, quinine 
should be withheld until the blood examination has been made, because 
the ingestion of doses of this drug, quite insufficient to influence the 
course of the infection, will often cause a disappearance of the para- 
sites from the circulating blood, for the young merozoites may be killed 
as soon as segmentation takes place, or the shape and staining reac- 
tion may be materially altered. 

Occasionally it is impossible to demonstrate parasites in the ordinary 
blood films after the most painstaking search, even when quinine has 
been withheld. In such instances, concentration of the blood film 
may be employed. 



92 EXAMINATION OF THE BLOOD 

According to the Ross-Ruge method, a very thick smear is made on 
a slide. After drying, the slide is treated with the following fluid : 

Formalin 5 cc. 

Acetic acid 1 cc. 

Distilled water, to make 100 cc. 

This serves to fix the smear and at the same time to take out the 
hemoglobin. The slides should be washed with water, dried and 
stained with Wright's stain, followed by treatment with borax-methyl- 
lene blue (borax, 2.5 gm.; methylene blue, 1 gm.; distilled water, 50 cc). 

This method is being supplanted by the concentration method of 
Bass and Johns: 10 cc. of blood is obtained by venipuncture and 
placed in a tube containing 0.2 cc. of a solution of glucose and sodium 
citrate (sodium citrate, 50 gm.; dextrose, 50 gm.; dissolved by heat with 
sufficient water to bring volume to 100 cc). The blood is mixed well 
and divided between two centrifuge tubes (preferably round bottom 
tubes 15 cm. inside by 12 cm. long), placed in Cornell shields. These 
tubes are centrifugalized at about 2500 revolutions per minute, long 
enough to bring the parasites and leukocytes to the top layer of the 
sediment and still not long enough to pack the corpuscles. It has been 
determined by Bass and Johns that if the radius of the centrifuge be 
18 cm., the time should be one minute for each cm. of blood column. 
At the end of the proper time interval, the top layer of sediment should 
contain all the parasites and the leukocytes. A capillary pipette is 
used to skim off this 1 mm. layer with an equal quantity of serum and 
to place the mixture in a tube about 12 cm. long and 0.5 cm. bore. 
The tubes may be made from glass tubing of suitable size. Should the 
column exceed 5 cm., the mixture should be divided between two tubes. 
The tubes are again centrifugalized, the top layer of sediment is drawn 
into a large capillary pipette to form a column not over 5 cm. in height 
which is mixed by forcing in and out on a slide, and is finally drawn 
back up into the capillary in such a position that the end may be sealed 
in the flame. The capillary is cut with a file above the blood column 
and carefully packed with a little cotton into a centrifuge tube so 
that it may be centrifugalized. When this has been done the gray 
leukocyte layer at the top of the sediment with the cells directly under- 
neath should be removed with a fine capillary pipette, placed on a 
slide, mixed, smeared in the usual way, and stained with Wright's 
stain. 

Development of the Parasite.— The understanding of the blood picture 
in malaria is simplified by knowledge of the life history of the infecting 
parasite. The organisms belong to the order hemosporidia, subclass 
telosporidia of the class protozoa. Ordinarily the infection of man 
takes place through the bite of an infected female Anopheles mosquito, 
which discharges through the veneno-salivary duct in the hypopharynx 
the sporozoites into the circulation of the human upon whose blood she 
is feeding. In this human intermediate host only the asexual cycle 



DISEASES IN WHICH THE BLOOD CONTAINS PARASITES 93 

of the development (schizogony) takes place. The sporozoite enters 
a red cell, grows into a schizont, when division of the chromatin takes 
place with the formation of a number of merozoites. These are liber- 
ated coincidently with the chill and infect a corresponding number of 
red cells. Each of these newly formed bodies repeat this cycle, and the 
multiplication of the parasites proceeds by geometrical progression. 1 

The sexual cycle (sporogony) takes place in the body of the Anopheles 
mosquito. When the mosquito has ingested blood from an infected 
human, the gametes are taken into the stomach and the macrogameto- 
cyte (female form) goes through the process of nuclear reduction with 
the formation of polar bodies. The microgametocytes (male form) 
extend rapidly motile flagellar projections which are broken off and 
become microgametes. One of these enters a macrogamete and fertil- 
izes it, an ookinete or vermicule resulting. This penetrates the wall 
of the stomach by a boring motion and lodges just under the outer 
layer of the midgut, where development proceeds into a large zygote 
or oocyst. The oocyst has a capsule and contains numerous small 
nuclear bodies. Each of these spherical bodies (sporoblasts) contains 
chromatin-bearing slender rods. The oocyst ultimately ruptures into 
the ccelomic cavity where the rod-like bodies (sporozoites) are dis- 
charged. They are carried from here by lymph currents to the salivary 
glands, from which they are discharged through the veneno-salivary 
duct when the Anopheles bites a new victim. 

The reader is referred to the diagrammatic sketch (Fig. 23) which 
illustrates the steps in development. 

The Study of the Stained Specimen.— In studying a stained specimen, 
care should be taken to prevent mistaken diagnosis. A frequent error 
on the part of students is to identify a platelet superimposed upon a 
red cell as a Plasmodium. This error should not be made if one has 
sufficient familiarity with the appearance of platelets. Furthermore, 
careful focusing will show the suspected body and the red cell to be in 
two different planes. Then the central portion of the red cell may show 
a markedly increased central pale area. This should not be taken for 
a parasite. In mounting a peculiar artefact is sometimes seen, giving 
the appearance of an air-bubble lying on the red cells. This is usually 
due to insufficient drying of the cover-slip before mounting. Though 
they do occur, mistakes should not be made on this score because 
of the refractility of the circular artefacts and their great number. 
Bits of leukocytic nuclear fragments might also cause confusion, which 
should be prevented by close scrutiny and adhering to the requirements 
of a definite and complete morphology before making a diagnosis. 

1 While the trend of opinion is that the life of the parasite is intracellular, Mary 
Rowley-Lawson has advanced evidence tending to confirm the view long held by some, 
that the parasite is extracellular during its whole life cycle. She asserts that it migrates 
from red corpuscle to red corpuscle, destroying each before it abandons it; that in the 
brief interval between, the parasite is free in the blood serum ; and that it almost imme- 
diately attaches itself to another red corpuscle by means of delicate pseudopodia. 



94 



EXAMINATION OF THE BLOOD 



Infection with Plasmodium Vivax (Benign Tertian Malaria).— Infec- 
tion with P. vivax is responsible for most instances of malaria seen in 
temperate and semitropical regions. The period of asexual develop- 




Sexual forms 

in man 

Fig. 23. — Diagrammatic representation of the life-history of the malarial parasite. 



ment consumes forty-eight hours, so that segmentation and accordingly 
a paroxysm occurs at forty-eight-hour intervals. A daily chill would 
be possible if the patient were bitten on two occasions, so that sporula- 



DISEASES IN WHICH THE BLOOD CONTAINS PARASITES 95 

tion could occur on alternating days. The blood taken shortly after 
a chill usually shows numerous young "hyaline bodies," merozoites, 
each occupying a red cell. These are tiny rings in one red cell. At the 
same time, the blood may show some older forms, since all the parasites 
do not develop absolutely simultaneously. As development proceeds, 
the body assumes an ameboid form or a looped figure, and at twenty- 
four hours occupies one-half or more of the infected cell, which is 
swollen to about one and a half times its usual size and is pale and 
anemic looking. The parasitic body is now termed a schizont. Fine 
brown pigment is scattered through the body of the parasite. 

The parasite continues to increase in size up to the end of the forty- 
eight-hour period when the pigment clumps at the center, the chromatin 
divides and from 15 to 20 small circular blue bodies are found, each 
with a bit of chromatin. This form is sometimes spoken of as a 
"rosette." After sporulation, there are set free from 15 to 20 sporo- 
zoites (hyaline bodies), each of which attacks and enters a new red 
blood cell. 

The gametes resemble the grown asexual forms, except that the ring 
is thicker while the chromatin is in one mass and the pigment is 
scattered. This is exactly opposite to the arrangement in the full 
grown asexual forms. The identification of asexual forms in the P. 
vivax infection may be difficult for a beginner and is not of especial 
practical importance. 

Infection with Plasmodium Malaria (Quartan Malaria).— The period 
for the completion of the asexual cycle is seventy-two hours, so that 
the chills occur at seventy-two-hour intervals. With a single quartan 
infection, there would then be two afebrile days between each day 
marked by paroxysm. If the patient were infected twice so that the 
blood contained two sets of parasites, sporulating on different days, 
there would be two febrile days, one day without chills and fever, again 
two febrile days, and so on. A number of combinations are possible, 
such as a quartan and a tertian infection. These possibilities suggest 
themselves to the reader. 

The parasite as seen in the stained preparation offers certain points 
of difference from P. vivax. The hyaline rings are thicker and the 
schizont (partly grown form) shows coarser pigment granules which 
are usually grouped peripherally about the edge of the slightly ameboid, 
oval parasite. The infected cell is usually somewhat shrunken. The 
presegmenting form is more symmetrical, the segmentation is into a 
few sporozoites (8 and 10), and the pigment arranges itself along the 
lines of division between the merozoites instead of at the center, as 
with P. vivax. 

Infection with Plasmodium Falciparum (Eslivo-autumnal Malaria).— 
With estivo-autumnal malaria, the paroxysms may occur every other 
day. The hyaline bodies are smaller and more delicate in outline. 
As development proceeds, the asexual forms disappear from the periph- 
eral circulation and one is able to find only a few hyaline bodies 



96 



EXAMINATION OF THE BLOOD 



and possibly the sexual forms (gametes). When only hyaline bodies 
are found on repeated examination, one's suspicions should be directed 
at once to the possibility of an estivo-autumnal infection. The 
gametes are characteristic of this infection, appearing as ovoid or 
crescentic bodies. In the case of the crescents, the red cell may be 
seen, appearing to fill in the space between the two rounded horns of 
the crescent. This is sometimes found in the "bib." The female 
gamete (macrogamete) is larger, more granular, does not give off 
flagella, the cell body stains more deeply, and the pigment surrounds 
a scanty and compact chromatin mass in the center. The male 
(microgametocyte) gives off flagella, is more delicate and hyaline, its 
entire body stains more faintly, the pigment is scattered throughout 
it, and chromatin is more abundant. 

Some workers (notably Craig) hold that there are two varieties of P. 
falciparum, the cycle with one being twenty-four hours, with the other 
forty-eight hours. 

Differentiation Between Types of Malarial Infection.— Clinically, the 
temperature curve should be considered, bearing in mind the fact 
that chills on alternating days may be produced by P. vivax or P. 
falciparum infection, and also the fact that the patient may be suffering 
with a multiple infection with one or more species of parasites. The 
following table summarizes the chief differential points found in 
examining the stained specimen. 

PRINCIPAL DIFFERENTIAL POINTS. 



Plasmodium vivax 
(benign tertian). 



Plasmodium malaria 
(quartan) . 



Plasmodium falciparum 
' (estivo-autumnal). 



Appearance of 
fected red cell 



Hyaline body 

(young schizont) 



Partly grown schi 
zont 



Pigment as seen in 
mature schizont 



Presegmenting 
form ' ' 



Sexual forms 



Larger size, lighter 
pink than normal 



Chromatin mass 
single 



Normal size 
staining 



Rings thicker 
round 



and 



and 



Distortions, some 
polychromatophilia 
and st rippling. 

Rings small and very 
fine; often 2 chro- 
matin dots. 



Chromatin in single j Body more marked, Seldom found in cir- 
mass; body with j extending as a culating blood, 
vacuoles or in loop- band across the 
like arrangement red cell 

Abundant fine pig- Coarse pigment ar- j Seldom found in cir- 
ment distributed '• ranged peripherally! culating blood, 
throughout para- 
sites 



Irregular division in- ; More regular divi- 
to fifteen or more i sion into ten or 
merozoites i less merozoites 

| Difficult to distin- Difficult to distin- 
guish in peripheral \ guish in peripheral 
blood from mature 
schizont | schizont 



Seldom found in cir- 
culating blood. 



Typical crescentic 
and ovoid bodies. 



DISEASES IN WHICH THE BLOOD CONTAINS PARASITES 97 

Summary of the Blood Changes.— The changes in the blood picture are 
summed up by Craig as follows : "A marked reduction in the number 
of red corpuscles, both by parasitic invasion and as a result of poisons 
elaborated by the plasmodia during their development, as well as 
changes brought about in the blood-forming glands by the malarial 
infection; a corresponding reduction in the number of white cells, with, 
in most cases, a relative increase in the large mononuclear leukocytes; 
a marked reduction in the hemoglobin, and the presence of black and 
brownish-yellow pigment, in greater or lesser amount." 





Fig. 24a 



Fig. 246 




Fig. 25 



Fig. 26 



Comparison of chief characteristics of Culex and Anopheles. Fig. 24a, the curved 
body of Culex when resting is approximately parallel to wall while the straight body 
of Anopheles. Fig. 24fe, stands out at an angle of about 45°. In Culex (Fig. 25) the 
palpse, of the female are short, while those of the male are longer than the prol 
In the Anopheles, the proboscis of both sexes are approximately equal in length. 



The Malarial Mosquito.— The Anophelinse may be distinguished from 
non-malarial mosquitoes by certain characteristic features. 

7 



98 EXAMINATION OF THE BLOOD 

The palpi of female Anophelinse are as large or larger than the 
proboscis. The palpi of the Culicidae are short and stubby. The 
males of both subfamilies have long, plume-like palpi. With the 
Anopheles, the proboscis is in the same straight line as the rest of the 
body, and in resting on a wall, the body projects almost at an angle 
of about 45°, supported by the first two pairs of legs, the last pair pro- 
jecting into the air. The Culex, however, rests in a "hunched back" 
position with the body bent. 

Cultivation of the Plasmodium.— The following method has been 
introduced by Bass and Johns. Blood is obtained by venipuncture 
under aseptic conditions and placed in a sterile tube, about one inch in 
diameter, and long enough to go in the centrifuge, containing 0.1 cc. 
of 50 per cent, glucose solution for each 10 cc. of blood. The blood is 




Fig. 27. — Trypanosoma gambiense showing the usual form; some cells in process of 
division. Magnification 1500 times. (From Novy.) 

defibrinated by stirring with a sterile glass rod. It may be kept in 
the same tube or transferred to other tubes. In either case, the column 
of blood must be one to two inches deep. There should be a column of 
serum at least § inch to 1 inch above the cells. The culture tubes 
should be incubated at 40° C. The parasites develop in the upper 
layer of precipitated cells. Red cells from this layer may be withdrawn 
with a sterile capillary pipette for staining and study. Cells from a 
lower layer contain only dead parasites. If more than one generation 
is desired, the leukocytes must be removed when the culture is made 
since they destroy the parasites at the time of segmentation. The 
freshly drawn blood is placed in a sterile tube, which will fit in the 
centrifuge. The centrifuge should be run at a speed of 800 to 2000 
revolutions per minute. The speed should be such that it will force 



DISEASES IN WHICH THE BLOOD CONTAINS PARASITES 99 

the red cells to the bottom of the tube and the leukocytes to a layer on 
top of the red cells. The exact length of time varies with the speed, 
the length of the centrifuge arms, etc., and must be learned by experi- 
ment. When centrifugalization has been carried out properly, the 
supernatant serum is drawn off and placed in culture tubes. Cells 
and parasites are now drawn with a pipette from the middle of the cell 
column and placed at the bottom of the culture tubes underneath the 
layer of serum. It is advantageous to employ flat bottom tubes. 

Trypanosomiasis.— This is a febrile disease, characterized by wast- 
ing, edema of the lower eyelids, an erythematous eruption, localized 
cyanosis, weakness and often lethargy, so-called "sleeping sickness." 
It is seen in South Africa and is due to infection with Trypanosoma 
gambiense (T. hominis, T. Castellani). The infection is carried by 
a species of tsetse fly, Glossina palpalis. A more virulent type of 
the disease is due to infection with the Trypanosoma rhodesiense, 
transmitted by Glossina morsitans (Stitt), is seen in South Central 




Fig. 28. — Filaria sanguinis. (Simon.) 

Africa. The trypanosom.es are blood flagellates, with a fusiform body, 
an undulating lateral membrane and two chromatin-staining areas. 
For diagnosis, peripheral blood may be used, preferably by a thick- 
smear method. Staining can be accomplished by Wright's stain, as 
for the malaria parasite (Fig. 27). Stitt recommends mixing about 
10 or 20 cc. of citrated salt solution, centrifugalizing two or three times, 
and examining the sediment after the last sedimentation, spreading it 
upon a slide and staining suitably. In severe cases the organisms 
may be discovered in the cerebrospinal fluid. Centrifugalization and 
examination of the sediment is desirable. 

Filariasis.— There are four principal species of the genus Filaria 
which produce the disease known as filariasis. They are Filaria 
bancrofti, Filaria loa, Filaria perstans, and Filaria medinensis. They 
belong to family filariidse of the class nematoda. Of these, Filaria 
bancrofti is the most common and it is the cause of more frequent 



100 EXAMINATION OF THE BLOOD 

manifestations, such as hematuria, chyluria, elephantiasis, and lym- 
phatic obstruction. 

The worm lives in the lymphatics of the trunk and extremities, the 
male and female being found together. The male is about 2 inches 
long and the female about 3 inches long. The embryos are found 
in the circulating blood, showing nocturnal periodicity. They are 
about 0.2 to 0.4 mm. long, and are sheathed in a gelatinous envelope. 
When stained, a number of nuclei are seen (Fig. 28). In examining 
the blood in suspected cases, the specimen should be secured, fixed by 
flooding with methyl alcohol for two minutes, and then stained with 
thionin or hematoxylin. It is both unnecessary and inadvisable to 
stain the red cells. 

Numerous reports have been made from time to time of cases of 
filariasis in the south. Johnston's work is interesting as an indication 
of the prevalence of the infection. He examined the blood of 400 
patients in the wards of a general hospital in Charleston, S. C, in the 
order of their admission and without regard to the condition for which 
they were admitted, and found that 19.25 per cent, harbored micro- 
filaria in their blood. In 1919 Edward Francis published a survey of 
nine other southern cities, and found comparatively few cases. He 
thinks that Charleston is an endemic focus because of the continued 
presence of many cases with microfilaria in the blood exposed to con- 
stant biting by Culex fatigans. 

For detecting the filaria in the blood, Johnston recommends the 
technic of Rivas and Smith: One cc. of blood is added to 9 cc. of 2 
per cent, acetic acid. This is shaken thoroughly and centrifugalized. 
The red cells are hemolyzed leaving only a small amount of sediment. 
The sediment is taken up with a capillary pipette and spread upon a 
slide for examination. While Rivas and Smith found parasites in all 
infected cases by using this method regardless of the time at which 
the blood was taken, they found the greatest number of parasites 
between 8 p.m. and 8 a.m., and between 8 a.m. and 8 p.m. in F. diurna. 

Filaria Loa (F. Diurna).— The embryos resemble F. bancrofti closely 
but have a diurnal periodicity. The adult worms tend to wander 
about in the subcutaneous tissue, especially that of the orbit. Infec- 
tion is common in South China, the Pacific Islands, the West Indies, 
and India. Occasional cases are seen in the United States, most of 
which are imported. The female worm, which is about 2 mm. in 
diameter and about 50 to 80 cm. long, is found in the lower extremity. 
The embryos are evidently swallowed in drinking water and migrate 
from the stomach to the lower extremity where the female burrows 
head down to the skin, until the head projects through the skin, an 
ulcer being formed at this point. The embryos are discharged through 
this ulcer, especially upon contact with water. Only the female worm 
was known until recently. The male is quite small and evidently dies 
and is discharged by the human' host. 



BACTERIOLOGY OF THE BLOOD 



101 



Relapsing Fever.— Spirochete infection is considered as the cause of 
the relapsing fevers, S. Duttoni causing the tick fever or relapsing 
fever found in East and West Africa and S. berbera causing that seen 
in North Africa and Egypt. The former is transmitted by the bite of 
an argasine tick, Ornithodorous moubata, and the latter by the bite 
of infected louse. The spirochetes may be demonstrated in the shed 
circulating blood (Fig. 29), either by the dark field illuminator or by 
staining dried films with Wright's stain. 




Fig. 29. — Spirochete in the blood. (Meader's case.) 

The report by Meader of five cases of relapsing fever in Colorado and 
the subsequent report of another case by Waring, which had been 
infected in the same region as had Meader's case, directs our attention 
to this infection. The carrier in these cases has not been determined. 



BACTERIOLOGY OF THE BLOOD. 

The technic of blood cultures is not difficult but the identification 
of the organisms which may be isolated requires training in bacterio- 
logical methods . It is unfortunate that bl ood cultures are not employed 
more extensively, since positive results are of great clinical value. 

Blood should be obtained by venipuncture in sufficient quantity, 
that is, about 10 to 20 cc. The practice of securing blood from the 
finger tip or the lobe of the ear is open to objection on two grounds. 
In the first place, it is impossible to secure a quantity which is neces- 
sary for the detection of the organisms when they are few in number. 
An even more urgent objection is the possibility of contamination. 



102 



EXAMINATION OF THE BLOOD 



Apparatus.— A syringe of the Luer type is an ideal instrument, since 
the form of construction does away with difficulties which arise when 
washers are used and when pistons are made to fit by wrapping them 
with some sort of packing. The syringe should be tested before 
sterilization with water to determine that it draws well and evenly, not 
with a jerky motion. The best safeguard is to purchase only syringes 
made by manufacturers of established reputation. 

The advantage about a syringe is that it may be used under practi- 
cally all circumstances. A number of other devices have been suggested. 
Stitt has proposed one which is described elsewhere (page 124). The 
writer has modified this somewhat (page 125). With either form of 
apparatus, the blood is drawn into normal salt solution containing 
sodium citrate or sodium oxalate. It must be transferred from this to 
the media into which it is to be inocu- 
lated. 

Especial pains should be taken to 
have a well-sharpened needle. Time 
spent in putting the needle in condition 
by sharpening the point and by rubbing 
off any rust which may have formed on 
the shaft will be well spent, since the 
puncture will be made much easier for 
the operator and much less painful for 
the patient. It will be found that a 
short needle will be easier to handle and 
that the blood will flow more readily if 
the gauge be moderately broad, about 
20 gauge. An irido-platinum needle is 
desirable, since it does not corrode, re- 
tains a sharp point well, and may be 
passed through the flame just before using 
without drawing the temper. 

The only other equipment required 
includes a piece of rubber tubing about 
two feet long for a tourniquet, alcohol lamp, sterile gauze or cotton, 
alcohol and tincture of iodine for sterilizing the skin, culture medium, 
and sterile pipette for transferring the mixture of blood and citrated 
salt solution to the media, if the blood be obtained in this way. 

The apparatus must be carefully sterilized. This may be accom- 
plished by autoclaving the syringe and needle or other bleeding appa- 
ratus at twenty pounds pressure for twenty minutes. It is convenient 
to place the barrel of the needle in one large test-tube, the plunger in 
another, and the needle in a third smaller tube, stoppering all with a 
well-fitting though not tight cotton stopper (Fig. 30). These may be 
autoclaved and carried readily. When they are used, the plunger 
should be taken out by the end of the handle and slipped into the 
barrel while the latter is still in its tube. The assembled syringe 




Fig. 30. — Sterilization of syringe 
(a and 6) and needle (c) in test- 
tubes for blood cultures. 



BACTERIOLOGY OF THE BLOOD 103 

may now be withdrawn, still handling it by the extreme end of the 
handle, and the tip may be fitted into the butt end of the needle which 
is in its test-tube, point down. The needle may be picked up in this 
way without touching it and the apparatus is ready for use. 

If more convenient, the apparatus may be boiled for fifteen minutes 
in 1 per cent, aqueous solution of sodium carbonate, or sterilized for 
an hour at 150° C. with dry heat. Boiling in ordinary water should 
not be depended upon for it may not kill spore-bearing organisms. 

Venipuncture.— The technic is described on page 126. 

The operator need use no precautions regarding the preparation 
of his hands except those which ordinary cleanliness demand, but 
should observe bacteriological technic rigidly, touching with his hands 
nothing that comes in direct contact with the blood. The syringe should 
be handled as one handles the platinum loop in the bacteriological 
laboratory, by the distal end only. - 

Inoculation of Media.— When the syringe is employed, the inoculation 
of the blood into the culture media must be made at once. Varying 
quantities, ranging from 1 to 3 cc. or even more may be ejected into 
flasks of sterile bouillon. The cotton stoppers should be removed and 
the mouths of the flasks passed through the flame first. After the 
blood has been added, the end of the cotton stopper which fits into the 
flask should be burnt in the flame before its insertion into the flask. 
When plates are made with agar or gelatin, the medium should have 
been liquified in boiling water and the tubes should have been placed 
in a tall receptacle containing water at 35° C. to cool the contents to a 
temperature of 40° C. The blood may be injected into the agar tubes 
from the syringe just as it was placed in the flasks, flaming the end of 
each tube carefully before adding the blood. With the tube held in a 
position as nearly horizontal as possible without letting the contents 
run out, it should be rolled as soon as the blood has been added to it 
between the palms of the hand to mix blood and medium thoroughly, 
the mouth is again flamed, and the contents are poured quickly into 
a Petri dish. In pouring, the cover should be lifted from the Petri 
dish as little as possible, to prevent air contamination. After the 
medium has thoroughly solidified, plates should be inverted, and the 
two portions should be held together with a strip of adhesive plaster or 
a broad elastic band. All the inoculated media should be placed in the 
thermostat at 37° C. at once. 

If either Stitt's or the writer's form of apparatus be employed and 
the blood be taken into citrated or oxalated salt solution, the con- 
tainer may be tightly stoppered with a sterile stopper and taken some 
distance to the laboratory or even sent by mail. It is preferable, 
however, to make the cultures into suitable medium as soon as possible. 
The citrated blood should be transferred with a sterile pipette to the 
culture medium and should not be poured. 

When the cultures have been made, they should be observed for a 
period of at least a week before being pronounced sterile. 



104 EXAMINATION OF THE BLOOD 

Types of Media.— The types of media to be employed will depend to 
some extent upon the infection which is suspected. For this reason 
if for no other one, the laboratory worker should be given enough of 
a knowledge of the case to provide suitable media. 

A good medium for general purposes is meat-infusion agar or meat- 
infusion broth which has been enriched by the addition of 2 per cent, 
of glucose. The same medium may be used without glucose, though 
its addition makes it suitable for a greater variety of organisms, 
particularly after the addition of human serum. 

Anaerobic cultures may be made by placing medium and blood in 
long tubes and by exhausting the oxygen in the usual way or by using 
Robinson's meat medium. They should be made when infection by 
the Bacillus aerogenes capsulatus or other anaerobic organisms is 
suspected. 

When typhoid or paratyphoid fevers are suspected, care should be 
taken not to have the media too highly acid. The use of an ox-bile 
medium has been recommended by many workers. This may be 
prepared by dissolving inspissated ox-bile (Merck's), 30.0 grams and 
peptone, 2.5 grams, in distilled water, 250 cc, and by autoclaving the 
solution in tubes. It is not necessary to use this medium, since 
typhoid bacilli will grow upon ordinary media, though bile has an 
inhibitory effect upon other contaminating organisms, and for that 
reason is preferred by those who take blood for cultural purposes from 
the finger tips or ears. When attempting to demonstrate typhoid 
group a relatively small amount of blood should be added to the 
bouillon flask, in order to avoid by the dilution the bactericidal effect 
of the blood serum. 

When one expects to find pneumococci or streptococci, it is well to 
use medium preferably containing glucose. This may be accomplished 
by adding to several flasks of the glucose-meat-infusion-broth which 
was recommended as a more or less universal medium, one or two grams 
of powdered calcium carbonate. Needless to say, this should be added 
before sterilization. Litmus milk also makes an excellent medium for 
the pneumococci. The exact directions for making media suitable for 
cultivating the pneumococcus are given in the chapter on Sputum 
(page 362). 

In attempting to isolate gonococcus from the circulating blood, it 
is well to have media which is neutral to phenolphthalein, and it is 
preferable to have it made from a veal infusion rather than from a 
beef infusion. Especial care should be taken to maintain the tempera- 
ture of the incubator at 37° C. Relatively large quantities of blood 
should be added. 

In order to permit growth at different gradients of oxygen tension, 
Rosenow recommends making shake-cultures in the columns of ascites- 
dextrose-agar. The blood is taken in the usual way and decalcified 
by placing it in a sterile container with 2 per cent, sodium citrate in 
normal salt solution. The proportion should be five volumes of blood 



BACTERIOLOGY OF THE BLOOD 105 

to one of the citrated salt solution. A portion of the blood is used for 
making cultures in flasks and in plates in the usual way. The remain- 
der is transferred by means of a sterile pipette to sterile distilled water, 
in the proportions of 1 cc. of citrated blood to 10 cc. sterile distilled 
water. The water should be in round bottom 50 cc. centrifuge tubes. 
These are centrifugalized and the sediment is removed with a sterile 
pipette and planted. The treatment with water serves to remove the 
hemoglobin and serum. 

The sediment is planted into long tubes containing a column from 
10 to 20 cm. high of dextrose-agar enriched with ascitic fluid. The 
agar should be 1 per cent, acid to phenolphthalein. The ascitic fluid 
should have been collected aseptically and should have been sterilized 
by heating at 60° C. for one hour on three successive days. One part 
of ascitic fluid is added to nine parts of agar after the agar has been 
boiled in a water-bath or steamed in an Arnold sterilizer for fifteen 
minutes to drive off the molecular oxygen. The heated agar should 
be cooled to 60° before adding the ascitic fluid. The medium is then 
cooled to 39° C. and the sediment from the laked sterile blood is added 
with a sterile capillary pipette, inserting the mouth of the pipette to 
the bottom of the tube containing the medium and forcing out the 
contents of the pipette as it is withdrawn. The cotton plug is replaced 
in the tube and the contents are mixed by partially inverting the tube 
several times. When planting the sediment, care should be taken not 
to blow any bubbles of air into the medium. Rosenow states that the 
space between the bottom and top of the tubes represents different 
gradients of oxygen, since the living cells in the bottom of the tube have 
oxygen-consuming power and so exhaust the oxygen, while the top 
remains aerobic. 

Colonies in the depth of the ascitic-dextrose-agar are examined by 
breaking the tube with a glass cutter and the red-hot end of a glass rod, 
flaming the surface of the tube just before breaking in order to sterilize 
it. The colony is fished out and suitable subcultures are made, repro- 
ducing in the subcultures as nearly as possible the conditions under 
which growth originally took place. 

To give directions for identifying the type of organism falls outside 
the scope of this work, except for such aid as may be secured in the 
section on simple bacteriological methods. If a growth appears sub- 
cultures should be made upon suitable media. If typhoid fever be 
suspected, and one finds a freely motile, Gram-negative bacillus, which 
fails to produce gas with glucose or lactose, the reasonable presumption 
is that it is the typhoid bacillus, but this must be confirmed by aggluti- 
nation tests (see pages 107 and 110). 

In cases of septicemia due to streptococcus and staphylococcus infec- 
tions, the percentage of positive results varies according to the site 
of the process. Probably the highest percentage of positive results is 
obtained with ulcerative endocarditis. Osteomyelitis frequently gives 
positive results in blood cultures. It is felt that the prognosis of 
staphylococcus infections is worse than streptococcus infections. 



106 EXAMINATION OF THE BLOOD 

The pneumococcus can be demonstrated in a definite proportion of 
cases, but the demonstration of the pneumococcus by blood culture 
cannot be said to have any reason for preference over the other clinical 
and laboratory methods which are more readily applicable. It was 
found by Avery, Chickering, Cole and Dochez that in cases in which 
a positive culture was obtained, the course of the infections was 
invariably severe and in many instances terminated fatally. 

The gonococcus has been reported in blood cultures. 

The list of other organisms reported as having been found is a fairly 
long one and serves to illustrate the number of infections which may be 
accompanied by at least a bacteriemia if not a septicemia. 

It has been pointed out by Libman, that the question of general 
infection is a variable one, depending upon the nature of the primary 
infection. For example, positive blood cultures are rarely obtained in 
uncomplicated erysipelas, in abscess of the lactating breast, in liver 
abscess, appendicitis, peritonitis of intestinal origin, pyelitis of preg- 
nancy, thrombosis of the pelvic veins, pyelophlebitis. He has also 
called attention to the fact that general infection as evidenced by blood 
culture is very uncommon with mastoiditis or otitis media, whereas 
positive blood cultures are obtained when the meninges or lateral 
sinuses are involved. In rheumatic fever the cultures are usually 
sterile except for the occasional finding of the Streptococcus hemo- 
lyticus, which does not seem to be the cause of the disease, since it is 
only occasionally present and since complement-fixation tests prove 
uniformly negative. 

The information furnished merely by bouillon cultures and the 
blood-agar plates gives much information regarding the streptococci. 
(For discussion of the use of blood agar for the study of streptococci, 
the reader is referred to the comprehensive work of James Howard 
Brown.) 

In giving consideration to the results of blood cultures, possible 
contamination should be carefully excluded. This should offer no great 
difficulty. If all the plates and flasks show a growth of the same 
organism, if the colonies are found deep in the agar and not merely 
on the surface, and if the organisms are of types ordinarily pathogenic 
and not such as might be expected from accidental contamination, it is 
reasonable to suppose that they had their source in the circulating 
blood. If, however, the colonies are found on one or two plates only, 
or in one or two flasks only, if they are on the surface of the plates, and 
if they are of varieties frequently found on the skin or in the air, the 
chances are strongly in favor of accidental contamination. Hiss and 
Zinsser's rule is excellent: "to look upon all staphylococcus blood 
cultures skeptically, discarding Staphylococcus albus as a contamina- 
tion, and taking, if possible, another corroborative culture when the 
organism is Staphylococcus aureus." 

Results of Blood Cultures. — In giving consideration to typhoid fever 
and in the paratyphoid infections, blood cultures are of great clinical 



SERUM REACTIONS 107 

assistance. Positive results are especially frequent in the first week of 
the disease, slightly less so in the second, markedly less so in the third. 
From this it may be seen that the results supplement those obtained 
by the Widal reaction, so that by using both methods it is possible to 
make a definite diagnosis on laboratory grounds in practically all 
cases. Gay states that when repeated blood cultures are made a 
positive diagnosis may be expected in from 75 to 85 per cent, of all 
cases of typhoid fever. The following figures, taken from Gay's work, 
are of interest: 

PERCENTAGE OF POSITIVE CULTURES IN RELATION TO TIME OF DISEASE. 













After 




1st week, 


2d week, 


3d week, 


4th week, 


4th week, 




per cent. 


per cent. 


per cent. 


per cent. 


per cent. 


Coleman and Buxton 


. 89 


73 


60 


38 


26 


Mann, Rainsford, Warren 


. 80 


62 


50 


50 




Gay 


. 73 


80 


53 


53 


33 



SERUM REACTIONS. 

Agglutination Tests.— The Widal Reaction.— The fact that infection 
by certain microorganisms results in the elaboration by the infected 
host of defensive bodies known as agglutinins, make it possible to 
examine the blood for these bodies as a diagnostic measure. For 
example, blood serum from a typhoid patient, even when considerably 
diluted, contains agglutinins, which will cause clumping or agglutina- 
tion of typhoid organisms. The tests based on this principle have 
assumed considerable clinical importance. The one for typhoid fever 
is called the "Widal" or "Gruber-Widal" reaction. The test is also 
applied to the diagnosis of the paratyphoid infections, and occasionally 
in dysentery and in cerebrospinal meningitis. 

The same technic is employed for performing the agglutination tests 
for typhoid and paratyphoid infections, the only difference, of course, 
being in the organisms which are employed. Either of two methods 
may be employed, each of which has certain objections and certain 
advantages, ^he microscopic method requires only small quantities 
of blood and it is completed a little more rapidly than the macroscopic 
method, which requires a greater amount of blood. The latter is the 
method of choice for scientific work, because of the ease with which 
accurate dilutions are made for the purpose of ascertaining the agglu- 
tination titer of the serum. 

Microscopic Method. — Stock cultures of the organisms to be employed 
for the tests must be at hand. These may be grown on solid media at 
room temperature and should be of known agglutinability. They 
should be transplanted once monthly. For performing the test, a 
subculture should be made either on an agar slant or in bouillon. 
The agar slant may be allowed to grow about twelve hours, the bouillon 
cultures about eighteen hours. The writer's personal preference is 



108 EXAMINATION OF THE BLOOD 

for the agar slant culture. If the bouillon culture be utilized, a drop 
may be mixed with a drop of diluted serum. When agar is preferred, 
2 or 3 cc. of normal saline solution should be poured into the tube, 
which is rolled between the palms a number of times until uniform 
suspension has been made of the organisms. A drop of the suspen- 
sion may be used for mixing with a drop of diluted serum. 

The only apparatus needed in addition to the microscope consists 
of hanging drop slides, i. e., object slides with a concavity ground out 
at the center (Fig. 31, a); vaseline or immersion oil for encircling the 
concavity in the slides; cover-slips; a platinum loop; a fine capillary 
pipette; a Wright's capsule or a small test-tube for collecting the blood; 
small watch-glasses, open salt dishes or small, flat staining dishes for 
mixing the serum to make dilutions. 

Normal salt solution should be at hand for making dilutions. 

Method.— Ywe or 10 drops of blood are obtained in a Wright's 
capsule (Fig. 31, b). If a blood culture is done on the patient at the 
time the blood is taken for the Widal reaction, the last half cc. of blood 




Fig. 31.— Apparatus required for microscopic agglutination tests, a, hanging drop 
slide; b, Wright's capsule; c, capillary pipette with rubber nipple. 

may be ejected into a small test-tube for the agglutination tests. The 
serum should be allowed to separate from the clot. This will usually 
take place spontaneously if the capsule is allowed to remain undis- 
turbed for ten or twelve hours. Separation may be accelerated by 
centrifugalization. If a capsule be employed, the end which is farther 
from the blood may be sealed in a low flame, taking care not to heat the 
blood, and after the glass has cooled, the capsule may be placed, sealed 
end down, in a centrifuge tube the bottom of which has been lightly 
packed with cotton. W 7 hen the serum has been separated, the capsule 
may be filed at or near the center and broken in two. A drop of 
serum should be placed in a watch-crystal marked A and a second drop 
into a crystal marked B. The pipette (Fig. 31, c) should be cleaned by 
drawing and ejecting successive changes of water, absolute alcohol, 
and ether. The residual ether should be thoroughly dried out by 
suction. It may then be rinsed with normal salt solution. After 
thorough cleansing, normal saline may be drawn into the pipette. 
Then nine drops should be placed in dish A and twenty-nine drops in 
dish B. A now contains a 1 : 10 dilution and B contains a 1 :30 dilution. 



SERUM REACTIONS 109 

Three hanging drop slides are taken and the edges of the concavity 
are ringed with vaseline or with immersion oil. On one cover-slip is 
placed with the platinum loop a drop of the 1 : 10 dilution of serum. 
To it is added with the platinum loop a drop of the suspension of 
typhoid bacilli, and the two drops are thoroughly mixed with gentle 
stirring, taking pains not to spatter any droplets onto the fingers or 
table top. The cover-slip is quickly inverted and mounted over the 
concavity of the hanging drop slide with the drop hanging down 
into the concavity which now becomes a sealed, moist chamber. The 
dilution is now 1:20. The dilution and time are written on the end 
of the slide with a grease pencil. The procedure is repeated with a 
drop of the 1:30 serum dilution, which becomes a 1:60 dilution after 
the addition of the drop of typhoid suspension. On the third slide 
should be similarly mounted a cover-slip upon which has been mixed 
a drop of the typhoid bacillus suspension and a drop of the normal 
saline solution used as a diluent. This serves as a control on the 
motility of the bacilli, and should show neither loss of motility nor 
spontaneous agglutination. 

The preparations should be examined at the end of thirty minutes 
and again at the end of an hour with regard to loss of motility and 
the clumping of the bacilli into groups of greater or lesser size. In a 
definitely positive reaction, there should be definite clumping and 
complete loss of motility; that is, there should be practically no un- 
clumped or motile organisms. The reaction is said to be doubtful 
when there are only a few clumps and only partial loss of motility. 
With a negative reaction the appearance of the slides containing 
serum is similar to that of the control slide, showing no loss of motility 
and no clumping. 

Use of Dry Blood.— The use of dry blood for performing agglutina- 
tion tests is not good practice, since it is impossible to make accurate 
dilutions. It is a method, however, which is employed considerably, 
especially in public health laboratories. It has the obvious advantage 
of making the sending of blood from a distance much easier. A drop 
of blood may be obtained from puncture of ear or finger and placed 
on a glass slide, a bit of aluminum, or even on glazed paper, dried, and 
sent to the laboratory. Upon its receipt in the laboratory, the dried 
blood may be rubbed up and dissolved with a little normal salt solution 
and transferred with the platinum loop to a cover-slip upon which has 
been placed a drop of the typhoid bacillus suspension. Dissolved 
blood should be added until the color of the mixture on the cover-slip 
is a delicate orange. This is assumed to represent a dilution of 1 :40. 

Precautions Against Infection. — Since living cultures of typhoid 
bacilli are being handled, great care should be taken to prevent any 
possibility of infection. The cover-slips on which the drops of serum 
and bacillary suspension are mixed should be held in cover-slip 
forceps or mounted on the end of a cork while the mixture is being 
made and under no circumstances should be held in the fingers. Even 



110 EXAMINATION OF THE BLOOD 

the patient's blood should be handled with care, since it may contain 
typhoid bacilli in the early stages of the diseases. The bacteria are 
not necessarily dead even after agglutination, therefore the cover-slips 
should be carefully lifted from the hanging drop slides with a pair of 
forceps, and the slides, cover-slips, and forceps should be dropped into 
a solution of phenol or lysol. The table top should be wiped up with 
one of these solutions. 

Macroscopic Method.— Fresh cultures are made on slants of agar, and 
after twenty-four hours' incubation, the growth is removed and a 
suspension made in sterile normal saline. This may be heated for one 
hour at 60°. At the end of this time cultures should be made to be 
certain that the suspension is sterile. The suspension may be kept 
in the refrigerator and should be well shaken before use. The suspen- 
sion must not be too thick. Experience will show the density which 
the emulsion should have. 

A suspension may be made according to the Oxford University 
method. The bacilli are grown for ten days or two weeks with daily 
subculturing. At the end of this time a few drops are placed into a 
larger Erlenmeyer flask, partly filled with veal infusion bouillon. The 
flask should be sterilized in the autoclave at 115° C. for no more than 
fifteen minutes and should be incubated before inoculation to make 
certain of its complete sterility. After inoculation growth is allowed 
to proceed at 37° C. for twenty-four hours, the flask is well shaken 
and for each liter of medium is added 1 cc. of commercial (40 per cent.) 
formalin. The contents are shaken and the flask is placed in the 
refrigerator at about 2° C. During the same day and on four or five 
subsequent days the flasks are shaken at intervals and replaced in 
the refrigerator. At the end of three or four days the bacilli will 
have been killed. Cultures should be made to determine absolutely 
the question of sterility. When sterilized, the culture may be made 
homogeneous by shaking in a mechanical shaking machine, or the 
larger particles may be removed by filtering through sterile cotton- 
wool. 

The apparatus required will include a small test-tube rack, seven 
small test-tubes, about 1 x 10 cm., accurately graduated volumetric 
Mohr pipettes (one with a capacity of 1 cc, graduated in T ^ cc), 
and an incubator or water-bath maintained at 37° C. Normal saline 
solution should be at hand. 

Seven small test-tubes are placed in the rack and 1 cc. of normal 
saline is placed in each tube, except the first, which receives 1.8 cc. 
In the first tube is placed 0.2 cc. of the serum to be tested. The serum 
remaining in the graduated 1 cc. pipette is discarded, and the serum- 
salt solution mixture in the first tube is drawn into and ejected out of 
the pipette to ensure complete mixing. The first tube now contains 
2 cc of a 1 : 10 dilution of serum. After thorough mixing, 1 cc. of 
this mixture is added to the salt solution in the second tube. The 
1 : 10 mixture remaining in the pipette after 1 cc has been measured 



SERUM REACTIONS 111 

into the second tube is ejected into the first tube and the pipette is 
then used for thoroughly mixing the contents of the second tube. 
After this has been accomplished, 1 cc. is transferred to the third tube. 
The residue is placed in the second tube, the contents of the third tube 
are thoroughly mixed, and 1 cc. of the mixture is carried to the fourth 
tube. This process is repeated until the sixth tube receives 1 cc. 
from the fifth tube. This is mixed as before, and then 1 cc. is discarded. 
Each tube now contains 1 cc. respectively of the following dilutions, 
beginning with the first; 1 : 10, 1 :20, 1 :40, 1 :80, 1 : 160, 1 :320. The 
seventh tube contains only 1 cc. of normal saline solution. To each 
tube is added 1 cc. of the bacterial suspension with a graduated volu- 
metric pipette. This results in doubling the dilution in each tube, 
the dilutions being respectively 1 :20, 1 :40, 1:80, 1: 160, 1 :320, 1 :640. 
The seventh tube now contains only normal saline and bacterial sus- 
pension, and serves as a control. After setting up the tubes in this 
manner, the dilutions should be marked on each tube with a grease 
pencil, the tubes should be shaken and placed in the incubator at 
37° C. for two hours. Then they may be allowed to stand at 
room temperature for six hours or in the ice-box over night, at the 
end of which time the readings may be taken. The control tube 
should show only uniform cloudiness. If a slight sediment is present, 
it should be possible to break it up with gentle shaking. With a positive 
reaction in the tubes containing diluted serum there will be a marked 
massing of the bacteria which will adhere to the sides and bottom of 
the tubes. 

If a mixed infection be suspected, the absorption agglutination test 
of Castellani may be performed; or preferably, agglutination tests 
according to Dreyer's method, testing the serum of the patient in 
parallel series of observations against standardized emulsions of the 
three microorganisms, B. typhosus and B. paratyphosus A and B, and 
determining the maximum dilution of serum in which agglutination 
takes place. For the technic of these, reference should be made to 
standard texts on immunology. 

Clinical Significance of the Results.— A positive Widal reaction with 
typhoid bacillus suspension and with a serum dilution of 1 :60 may be 
taken as evidence of a typhoid infection. While certain other diseases 
may give partial reactions with lower dilutions, and while even normal 
serum may clump the bacilli in a 1 : 10 dilution, it is generally agreed 
that only typhoid infections will-give a positive reaction with a dilution 
as high as 1:60. 

The exact time during the infection at which the patient's serum will 
first give a positive reaction is variable. While considerable work 
has been done upon the subject, it is difficult to correlate the results 
because of the fact that different investigators have used different 
dilutions, and because no uniform method has been adopted for 
determination of the day of onset of the disease. 

Approximately 80 per cent, of the cases give a positive result in the 



112 EXAMINATION OF THE BLOOD 

second week of the infection with a 1:60 dilution, taking as the first 
day of the disease the day when the patient took to his bed. The 
percentage of positive results increases somewhat with the progress of 
the disease and it is stated that if repeated examinations are made 
frequently enough during the course of the patient's illness, a positive 
reaction will be secured in about 90 to 95 per cent, of cases. The 
following figures have been reported as giving the percentage of 
positive results at different stages of the infection: 

PERCENTAGE OF POSITIVE AGGLUTINATION TESTS IN DIFFERENT 
PERIODS OF TYPHOID FEVER. 

1st week, 2d week, 3d week, 4th week, 5th week. Average, 
per cent, per cent, per cent, per cent, per cent. per cent. 

Park and Williams . . 20 60 80.0 90 ... 88.0 

Gay 61 82 87.5 92 100 91.8 

Frequently, the most marked agglutination is obtained during 
convalescence, and a positive reaction may persist for a period of weeks 
or months. Cases are reported where a positive Widal has been secured 
years after an attack of the disease. In such instances, the individual 
should be suspected of being a carrier. It is to be remembered that 
the inoculation of typhoid vaccines will produce a positive Widal and 
that this may persist for many months. That a negative reaction 
cannot be taken as sufficient reason for excluding the presence of 
typhoid should go without saying. 

Another factor which may cause confusion is the group agglutination 
that occasionally occurs with paratyphoid infections. A typhoid 
patient may give a partially positive agglutination with typhoid 
bacilli and one almost equally positive with paratyphoid bacilli. In 
such cases, the absorption method of Castellani would have to be 
utilized to clarify the diagnosis. 

Isohemagglutinins in Human Serum.— The presence of isohemagglu- 
tinins in human sera and the division of individuals into four groups 
according to agglutination reactions, makes the determination of the 
group to which donor and recipient belong of much importance when 
transfusion or skin grafting is contemplated. A donor for transfusion 
must belong to a group whose cells are not agglutinated by the recipient's 
serum. Severe and even fatal reactions may follow the injection 
of improperly selected blood. That the division into groups must be 
considered in skin grafting also has been shown by Shawan's work. 
The procedure is referred to as "blood grouping" or "blood matching." 

Jansky and Moss has found that all bloods may be divided into four 
classes. An individual retains his group characteristics throughout 
his lifetime. 

In Group I the serum does not contain any iso-agglutinins, and 
therefore does not agglutinate the corpuscles of individuals from the 
same or any other group. 



SERUM REACTIONS 113 

In Group II the serum contains an agglutinin known as agglutinin A. 
It will cause clumping of the cells of individuals from Groups I and III. 

In Group III the serum contains an agglutinin termed agglutinin B. 
It will agglutinate cells from Groups I and II. 

In Group IV the serum contains both agglutinins A and B. It 
clumps the blood corpuscles from Groups I, II, and III. 

The interactions may be represented as follows: 

No serum agglutinates its own cells. 

Group I serum agglutinates no cells. 

Group II serum agglutinates cells from Groups I and III. 

Group III serum agglutinates cells from Groups I and II. 

Group IV serum agglutinates cells from Groups I, II, and III. 

Group I cells are agglutinated by serum from Groups II, III, and IV. 

Group II cells are agglutinated by serum from Groups III and IV. 

Group III cells are agglutinated by serum from Groups II and IV. 

Group IV cells are not agglutinated by any serum. 

The group divisions among the population is approximately as 
follows: 

Group I comprises 7 per cent. 

Group II comprises 40 per cent. 

Group III comprises 10 per cent. 

Group IV comprises 43 per cent. 

While the grouping of Moss has been accepted generally heretofore, 
there is an increasing tendency to adopt that of Jansky, because of 
priority. A serum which would be classified as of Group I according 
to Moss is placed in Group IV by Jansky. Groups II and III are the 
same with both classifications. 

Lee's Method.— The apparatus required includes a hand lens, hanging 
drop slides, small test-tubes, and platinum loop. 

The reagents needed are normal saline solution and a small amount of 
sterile serum from Groups II and III. Karsner and Koeckert have 
shown that this serum may be preserved very simply for a period of 
months without appreciable loss in its agglutinin titer if placed in small 
capillary pipettes, hermetically sealed at both ends. When serum is 
kept in a dried state on glass cover-slips or filter paper, the titer is 
reduced and non-specific agglutination is seen. Before employing 
serum for tests, its power of agglutination should be demonstrated. 
For example, when one part of Group II serum is diluted with nine 
parts of sterile normal salt solution, it should agglutinate Group III 
cells. 

Blood is obtained from the patient (recipient) and from the proposed 
donors. In each case the blood is obtained from a puncture of the 
ear lobe. One drop is allowed to fall into a small test-tube previously 
charged with 1.0 cc. normal saline solution. The tubes are shaken very 
gently. One drop of each suspension is mixed with a drop of Group II 
serum and one drop is mixed with a drop of Group III serum. The 
platinum loop may be used for measuring the drops and for careful and 



114 EXAMINATION OF THE BLOOD 

gentle mixing. The results are to be observed under the low power of 
the microscope or with a hand lens at the end of five minutes. When 
agglutination is present, the cells will be seen clumped closely; while 
separation of the cells through the major portion of the drop is evidence 
of the absence of agglutination. 

The group may be determined with the aid of this chart, a plus sign 
representing the clumping of corpuscles, a minus sign representing the 
reverse. 

Group II serum. Group III serum. 

Effect upon cells of Group I + + 

Effect upon cells of Group II — + 

Effect upon cells of Group III + — 

Effect upon cells of Group IV — — 

For example, if clumping occurs with both drops of cell emulsion 
from a given blood, the individual who furnished the cells belongs to 
Group I; if clumping occurs with neither drop, the individual belongs 
to Group IV; if clumping is seen with the drop mixed with Group II 
serum and not with the drop which was mixed with Group III serum, 
the individual belongs to Group III, and conversely, if the agglutina- 
tion occurs with Group III serum and not with Group II serum, the 
individual belongs to Group II. In performing tests, it is advisable 
to set up a control with a known normal blood. 

If Jansky's classification be followed, the following table illustrates 
the effect of sera of Groups II and III upon the cells of the four groups. 

Group II serum. Group III serum. 

Effect upon cells of Group I — — 

Effect upon cells of Group II — + 

Effect upon cells of Group III + — 

Effect upon cells of Group IV + + 

Application of Results.— In transfusions or skin grafting it is ideal 
to have recipient and donor belong to the same group. This is not 
always possible, however, and a donor may be used whose cells are not 
agglutinated by the recipient's serum. 

Rons and Turner's Method.— This method has the advantage that 
known sera are unnecessary since the blood of the recipient is tested 
against that of the proposed donors. Rinse the 1 : 10 hemocytometer 
pipette with 10 per cent, aqueous solution of sodium citrate, and draw 
citrate solution to the 1 mark. Fill pipette rapidly with blood from 
puncture of patient's ear or finger, and expel mixture into a small, 
narrow test-tube. Repeat operation with each of the prospective 
donors. If several donors are to be tested, 2 or 3 pipettefuls of blood 
should be taken from the patient. 

The mixing of the patient's blood with that of the donor's is done in 
Wright's pipettes (Fig. 31 C), drawing blood to the mark, followed by a 
small amount of air, and then another portion of blood, repeating the 
operation until the desired dilution has been obtained. With the 



SERUM REACTIONS 115 

blood of each donor, two mixtures are made, one containing 9 parts of 
patient's blood to 1 of the donor's, and one containing equal parts. 
The dilutions are ejected on a microscopic slide and drawn well up into 
the pipette to ensure mixing. After fifteen minutes the sealed end is 
broken and a small drop of the contents expelled on a slide, super- 
imposing a drop of salt solution, and covering with a cover-slip. Readings 
should be made with the microscope, noting the presence or absence 
of agglutinated clumps. When present, these are usually of fairly 
uniform size in a given preparation and are sometimes large enough to 
be macroscopically visible. 

If clumping be present in the 1+9 mixture and to a less degree or not- 
at all in the 1+1 mixture, the patient's blood agglutinates that of the 
donor and may hemolyze it, and the proposed donor cannot be used for 
transfusion. Clumping with 1 + 1 mixture and none with the 1+9 
mixture indicates that the prospective donor's blood agglutinates the 
patient's cells and the advisability of transfusion is doubtful. When 
there is no clumping in either mixture, the assumption is justifiable 
that the two bloods do not hemolyze or agglutinate each other and that 
transfusion is safe. 

Precipitin Reactions for Human Blood.— Animals which have received 
injections of a foreign protein (antigen) develop among other sub- 
stances an antibody for this antigen which, is termed precipitin. If 
serum containing the precipitin be mixed with protein of the sort used 
for antigen, a precipitate will be formed. This reaction has been 
taken advantage of in identifying the source of blood and other stains 
and has been found especially useful for medico-legal purposes. The 
reactions are not absolutely specific, since an antiserum obtained by 
injecting human serum into a rabbit will give a precipitation when 
mixed with cognate species of primates, especially the higher groups 
of apes, although the result will not be obtained with as high dilution 
as with human serum. 

Blood stains upon cloth, metallic bodies, glass and other objects 
may be subjected to the test. Stains of great age have given satis- 
factory tests. The dried blood may be scraped from a hard surface 
and dissolved with normal salt solution. If the stain' be on a piece 
of fabric, a portion should be cut out, carefully teased out with teasing 
needles or cut into fine bits with a pair of clean scissors, handling 
the fabric only with forceps to avoid the use of the hands, and the 
bits placed into a thoroughly clean test-tube. A small quantity of 
0.85 per cent, sodium chloride solution should be added, and the bits 
of fabric should be allowed to soak for a period of several hours. The 
process may be allowed to proceed in the incubator at a temperature 
of 37° C. for the first few hours and then allowed to continue at room 
temperature as long as twenty-four hours, if necessary. After extrac- 
tion, the solution may be filtered through fine filter paper, or even 
through a Berkefeld filter if necessary, to render it entirely clear. If 
the solution be not neutral to litmus, it should be made so by the 



116 EXAMINATION OF THE BLOOD 

addition of a dilute solution of either sodium bicarbonate or tartaric 
acid, as the case may be. 

The extract must be diluted with normal saline solution to the 
proper strength, approximately 1 : 1000. Solutions of the proper 
strength will show a slight foam when shaken, and they will give only 
a slight opalescence when the albumin test is done by heating a little 
in a tube and adding a drop of 25 per cent, nitric acid. The latter test 
also serves to show that the stain was made by a protein-containing 
substance. 

Before performing the precipitin test, it will be well to ascertain 
that the suspected stain is really blood by applying Teichmann's 
hemin crystal test. The benzidine test may be done also, but it is not 
regarded as satisfactory for forensic purposes. 

Preparation of the Antiserum. — Several rabbits or birds should be 
immunized, since it is difficult to secure an antibody of sufficiently 
high titer. Large animals should be used. Three injections may be 
given intravenously at daily intervals, giving 5 cc, 10 cc, and then 
15 cc. of sterile human serum. The serum may be titrated ten or 
twelve days later, and the animal bled after fasting, if precipitation be 
given by a 1 : 10,000 dilution. Another method is to give intravenous 
injections at intervals of five days, giving first 10 cc. then 8 cc, and 
next 5 cc. Titration of serum is done a few days after the last dose, 
and the animal is bled if it gives a high titer. If not, further injections 
may be given, although the mortality is high after the third injection. 
The animals may be immunized with intraperitoneal injections, but 
larger doses will have to be employed. The injections may be given 
and the blood secured for preliminary titration in the manner described 
elsewhere (pages 131 and 133). 

AYhen the results of the titer are satisfactory, the animal may be 
bled (see page 135) and the serum separated and stored in 1 cc. lots 
in hermetically sealed glass ampoules, under sterile precautions. The 
serum should be absolutely clear. If not, it should be filtered through 
a sterile Berkefeld filter before sealing for storage. 

Titration of the Antiserum. — The immune serum must be of such 
potency that a prompt precipitation will be produced when 0.1 cc. of it 
is placed in a test-tube and 1 cc. of human serum is floated over it 
with a pipette. This test should be carried out as one carries out 
the Heller's test for albumin. A fine white ring should form at the 
line of junction of the two fluids. 

Method of Performing the Test.— For the reaction slender test-tubes 
should be employed, preferably about 3 mm. in diameter and 10 cm. 
long. They should be carefully cleaned and should be free from any 
chemical impurity, although bacteriological sterility is not essential. 
Six tubes should be set up in a rack and suitably numbered. Controls 
must be used to demonstrate the fact that the antiserum will produce 
precipitation in the presence of human serum, that the unknown will 
not produce a precipitate with normal rabbit serum, and that neither 



SERUM REACTIONS 117 

the antiserum nor the unknown will form a precipitate with the salt 
solution employed in the test. Furthermore, when the stain has 
been extracted from cloth or paper a bit of the same material without 
stain should be extracted with normal saline solution and treated with 
the antiserum, since certain chemicals used in dyeing might give a 
precipitate. The following protocol serves as a guide to setting up 
the test and its necessary controls : 

Tube 1. 0.1 cc. antiserum; 1.0 cc. unknown solution to be tested. 

Tube 2. 0.1 cc. antiserum; 1.0 cc. known human serum, 1:100 
dilution. 

Tube 3. 0.1 cc. normal rabbit serum; 1.0 cc. unknown solution to be 
tested. 

Tube 4. 0.1 cc. normal saline solution; 1.0 cc. unknown solution to 
be tested. 

Tube 5. 0.1 cc. antiserum; 1.0 cc. normal saline solution. 

Tube 6. 0.1 cc. antiserum; 1.0 cc. normal saline extract of same 
fabric (when stain is on fabric) . 

The readings for medico-legal work should be taken in twenty 
minutes. The results in the first tube will depend upon the presence 
or absence of human blood in the suspected stain. A precipitate will 
appear if blood were the cause of the stain and no precipitate should be 
seen if blood were not present. The second tube is a control on the 
potency of the antiserum and should show a precipitate. No precipi- 
tate should appear in tubes 3, 4, 5, or 6. 

Sources of Error.— The substance on which the stain is found may 
interfere with the test. With stains on leather the conditions are 
unfavorable, since tannin or other substances used in tanning may 
cause precipitation of serum. Stains on earth are unsatisfactory. 
The lime which is usually present will have to be removed from the 
solution by precipitation with a current of carbon dioxide and filtration. 
It is to be remembered that the reaction will be given by higher species 
of apes, though if the proper dilutions are made possibility of confusion 
is reduced. Care should be taken definitely to establish the fact that 
the stain is due to blood, since any body secretion containing serum 
will give the reaction. 

Precipitin Reactions for the Detection of Other Proteins.— The same 
principle may be applied in establishing the species from which 
unknown proteins come. The reaction is utilized, for example, in 
determining whether a given sample of meat is admixtures of beef with 
pork instead of being pure beef or whether so-called sausage contains 
game or not. The antisera would be obtained by the injection into 
rabbit's blood from the species whose sera are to be tested. The 
technic would be the same as has been outlined. If a specimen of meat 
were to be examined, care should be taken to secure the specimen from 
the central portion, to cut it into small pieces with a sterile knife, and 
to mince it thoroughly with a perfectly clean chopping knife or meat 
grinder. An infusion should be made with normal saline solution for 



118 EXAMINATION OF THE BLOOD 

a period of about six hours at room temperature. The concentration 
of the extract should be adjusted so that it will be sufficient to produce 
a foam when shaken, but not strong enough to produce more than an 
opalescence when tested with heat and 25 per cent, nitric acid, as 
directed in the preceding section. If the substance is fatty, the fat 
should be extracted by letting the specimen stand in an Erlenmeyer 
flask over night covered with a mixture of equal parts of chloroform and 
ether. In the morning the ether-chloroform mixture is poured off, the 
meat is washed with normal saline, and extraction with normal saline 
is carried out as previously directed. 

The titration of the antiserum and the actual setting up of the test 
should be carried out as directed in the preceding section. 

Complement-fixation Reactions.— In regard to the group of com- 
plement-fixation reactions, certain things may be said in common. 
They are all time-consuming and no method has been devised for 
materially reducing the required time and labor without a simultaneous 
sacrifice of reliability in the result; the hemolytic system requires pains- 
taking attention to quantitative methods and a correspondingly 
accurate technic; the greater the number of specimens examined at a 
time with one set of reagents adjusted each to the other, the more 
dependable will be the results. The biological reagents which are 
required are not prepared without considerable labor and do not keep 
indefinitely. Unfortunately it has not been possible to simplify the 
technic so that any of the dependable complement-fixation reactions 
may be done unless time, adequate equipment, control sera, and train- 
ing be available. The dependability of the results will vary pro- 
portionately with the experience of the operator, his attention to minute 
and painstaking detail, the accuracy of his technic, the number of 
reactions which he is doing. 1 

Principle Underlying Complement-fixation Reactions.— Before proceed- 
ing with diagnostic serologic work, a preliminary knowledge of the 
fundamental principles of hemolysis and immunology is essential. 
The student should have a laboratory course such as outlined in the 
excellent manual of Zinsser, Hopkins and Ottenberg, "Laboratory 
Course in Serum Study," supplemented by study of reference texts, 
such as Kolmer's "Infection, Immunity and Specific Therapy," or 
Zinsser's " Infection and Resistance." 

The underlying principle is the fixation of the complement by the 
antigen through an amboceptor. The complement is present in all 
sera. The antigen is the inciting cause of the disease, the amboceptor 
is a specific defensive substance (antibody) elaborated by the body in 
response to invasion of the antigen. In the Wassermann reaction, 
however, the antigens employed are not prepared from the exciting 
cause of syphilitic infection (Treponema pallidum) but were at first 
extracts of organs presumed to be rich in treponemata, of the liver of a 

1 The list of apparatus needed and the directions for cleaning glassware are given in 
the Appendix. 



SERUM REACTIONS 119 

syphilitic fetus, etc. It was found that extracts of normal organs, 
fractions of these extracts, or extracts reinforced by cholesterol gave 
excellent and apparently specific results when checked with the clinical 
diagnosis. Antigens prepared in this way give a much higher per- 
centage of positive results in known cases of syphilis than do antigens 
made from pure cultures of treponemata. Therefore, the Wassermann 
reaction as performed at present does not employ a specific antigen 
and rests on empiric grounds, which clinical experience has shown to be 
quite secure. The complement-fixation reaction for gonorrhea and 
tuberculosis employ specific antigens and rest theoretically on a secure 
basis, but the practical results in tuberculosis are vastly less satis- 
factory. 

In the actual performance of any of the tests of this group, the 
reaction produces no visible change though we may place together 
antigen, complement and the patient's serum containing antibody and 
though fixation of the complement may take place. It is necessary 
then to employ some means of determining whether or not fixation 
occurred. For this purpose the so-called " indicator" or "hemolytic" 
system are used. After time has been allowed for the fixation of the 
complement, there is added to the tubes, red blood cells of a given 
species and hemolytic amboceptor. The latter is serum from a second 
species which has been immunized to the cells of the first. A number 
of hemolytic systems are used but in this manual we employ sheep 
cells and serum of rabbits immunized against sheep cells. In the 
"indicator system" the sheep cells are the antigen, and the rabbit serum 
contains the antibody or amboceptor. The complement is obtained 
from that originally placed in the tube, if any be still available. After 
treatment at suitable temperature, the "indicator system" shows either 
hemolysis, partial hemolysis, or no hemolysis, results which are clearly 
visible. The nature of the result will depend upon the availability 
of complement, and this in turn upon whether or not it has been fixed 
by the syphilitic antigen in the presence of a syphilitic antibody. For 
example, complete hemolysis (solution of the red cells) means that 
complement was still available, that this had not been fixed by the 
antigen of the first system and therefore that there was no specific 
antibody in the patient's serum. On the other hand, lack of hemolysis 
(no solution of the red cells) means that no complement was available, 
that it was fixed by the antigen of the first system, and that there was 
a specific antibody present in the patient's serum to effect this fixation. 
While the reaction may seem simple enough in theory, several factors 
have to be considered in its practical application. The quantitative 
relationship of the hemolytic or indicator system must be nicely 
balanced; the amount of the antigen employed must be sufficient to 
fix the complement in the presence of syphilitic antibody but not great 
enough to give non-specific fixation; the human serum must be freed 
from the complement which it contains to avoid adding additional 
complement, and also from anticomplementary substances; and sera 



120 EXAMINATION OF THE BLOOD 

must be devoid of marked bacterial contamination since this may give 
rise to a falsely positive result. These points, which must be safe- 
guarded technically, will be discussed in detail in the description of 
the method. 

THE WASSERMANN REACTION. 

General Considerations and Comparison of Methods. — Indicator System. 
—The original technic of Wassermann, Neisser and Bruck has many 
modifications, and one who begins this work is forced to make choice 
between them. Various indicator or hemolytic systems have been 
suggested. The two which have the most popularity are the one 
utilizing sheep corpuscles (as used in the original technic) and the one 
which employs human corpuscles and the hemolytic serum of a rabbit 
immunized against human corpuscles (as suggested by Noguchi for 
his modification). The method here described provides for the use of 
the first-named system. It is preferred because of the general avail- 
ability of sheep's blood and the ease with which hemolytic amboceptor 
is developed in rabbits. An antihuman hemolytic amboceptor of cor- 
responding strength is elaborated with much greater difficulty, and 
the mortality among rabbits subjected to immunization against human 
cells is high. The system employing sheep cells has one drawback, 
the presence in many human sera of a native hemolytic amboceptor 
for sheep corpuscles. In the method given here we attempt to obviate 
this objection, at least partially, by adding no rabbit serum hemolytic 
amboceptor to serum which contains native antisheep amboceptor, 
and by accurate titration of complement which, as Ecker and Sasano 
have shown, reduces the possibility of error even if excess of amboceptor 
be present. (In the writer's opinion the best method for performing 
the test with human cells is the method described by Craig in "The 
Wassermann Test," C. V. Mosby Co., St. Louis, 1918.) 

InacUvation of Human Serum.— The human serum to be tested is 
inactivated, that is, subjected to a temperature of 56° C. for thirty 
minutes to destroy the thermolabile complement and certain anti- 
complementary substances. Omission of this step has been urged, 
especially by Noguchi, chiefly on the ground that a portion of the 
specific antibody may be destroyed. However, most workers think 
that it is better to run the risk of destroying some of the specific anti- 
body rather than the greater one of adding unknown quantities of 
complement and of introducing anticomplementary substances. This 
is particularly necessary where cholesterol ized antigens are employed. 

^Intigens.—As has been stated, the antigens employed in the Wasser- 
mann reaction are really not specific. A number of types are now in 
general use, including pure alcoholic extracts of heart (human, guinea- 
pig, or beef), the same alcoholic extracts reinforced by the addition 
of cholesterin, or the lipoid fraction of these alcoholic extracts as 
recommended by Noguchi. The author's personal preference is for 
the cholesterol-reinforced alcoholic extract antigens, because of avail- 
ability, delicacy, uniformity and the ease with which they may be pre- 



THE WASSERMANN REACTION 121 

pared. Considerable controversy has been waged about the reliability 
of various types of antigen. This will be discussed in a consideration 
of the interpretation of results. 

Temperature for Fixation of Complement— The temperature which is 
used to accelerate the fixation of the complement by the antigen and 
specific antibody is also a point on which workers vary. The tempera- 
ture originally employed was 37° C, for one hour. This was shortened 
by some to thirty minutes when treatment by immersion in water-bath 
supplanted treatment by dry heat. Later fixation in the ice-box for a 
period of from four to ten hours was advocated. 

From the researches of several investigators, it would appear that 
cold fixation (i. e., permitting the fixation of the complement by antigen 
in the presence of the syphilitic serum at 8 to 12° C. from four to ten 
hours) gives a much higher percentage of results than fixation for one 
hour at 37° C. The explanation has been advanced that more complete 
fixation of complement takes place during the longer time-interval, 
and that exposure to ice-box temperature for this period of time is 
possible while exposure to body temperature would result in marked 
deterioration of complement. Smith and MacNeal have shown that- 
cold fixation enhances the action of cholesterolized and plain alcoholic 
extract antigens more than it does that of the acetone-insoluble anti- 
gens. Ottenberg found discrepancies between the two methods in 
15' of 120 cases in which parallel sets of reactions were performed, the 
greater number of positive results being obtained by the ice-box 
method. Of the 15 cases, 7 were clearly syphilitic, 2 were treated 
syphilitics, and 4 were probably syphilitic. 

Quantities Employed.— The quantities prescribed in the original 
technic were much larger than are used by most workers today, the 
total bulk of the contents of a reaction tube being 5 cc, using 1 cc. of a 
Yo dilution of complement. This was extremely wasteful of reagents, 
and the amount of the reagents has been reduced to fractions of these 
quantities in practically all laboratories. The exact bulk is immaterial 
as long as it is large enough to permit accurate measurement. Any 
method, however, which does not demand the accurate volumetric 
measurement of all reagents, including the serum to be tested, is to be 
severely condemned and the writer does not feel that the use of capillary 
pipettes for measurement fulfils the fundamental requirement of 
accuracy. There is a great variation in the bore of capillary pipettes 
as drawn by the average worker, so that the size of the drop delivered 
varies greatly and a tremendous error is introduced into a reaction 
where quantitative adjustment of the reagents to each other is abso- 
lutely essential. 

Modifications.— Many modifications of the Wassermann reaction 
have been proposed. As may be seen, there are a number of variable 
factors, i. e., the balance of the hemolytic system, the number of units of 
amboceptor and complement which are employed, the type of antigen, 
the strength of the blood-cell suspension, and the temperature at which 
fixation of the complement is allowed to proceed. 



122 EXAMINATION OF THE BLOOD 

Noguchi attempted to simplify the technic so that the reagents 
could be prepared in a central laboratory, and the test might be 
performed by the practitioner with slight laboratory facilities. He 
substituted a human blood-cell system for the sheep blood-cell system 
of the original Wassermann technfc, and proposed a scheme for the 
preservation of the amboceptor, complement and antigen in the dried 
form on paper. An antigen made up of acetone insoluble lipoids 
was devised, and the use of fresh human serum was recommended. 
The measurement of the reagents in the liquid form by the use of 
capillary pipettes was advocated. Certain objections to this method 
may be made. The preservation of antigen and complement on paper 
has not been found practical on account of rapid deterioration. It is 
much more difficult to secure an active antihuman than an antisheep 
amboceptor of high titer. The use of capillary pipettes as a means of 
accurate quantitative measurement is open to the criticism which has 
been made. Fresh human serum contains an unknown amount of 
complement, introducing a variable factor into the reaction. 

Craig modified the original technic by substituting an antihuman 
system for an antisheep system, making no other marked changes. 
His method is carefully worked out and should commend itself to one 
who wishes to employ the antihuman system. 

Bauer proposed employing the native antisheep amboceptor present 
in human sera. The presence of antisheep amboceptor, however, is 
too variable to permit one to rely upon it solely for carrying out the 
reaction. 

The modification of Hecht-Weinberg was based upon utilizing the 
native complement in human sera. Both of these are extremely 
variable quantities, but Gradwohl has devised a method for determining 
the hemolytic strength of a given serum by titration, and has modified 
the Hecht-Weinberg. Even though this reaction is delicate, Kolmer 
has stated that it is open to the same error that may occur whenever a 
crude alcoholic organic extract antigen is used with an active serum, 
i. e., the appearance of false positive reactions. 

Securing Specimens of Human Blcod.— It is preferable to secure this by 
venipuncture. To obtain enough from the finger or the ear for exami- 
nation by this technic is difficult, though it may be done. Specimens 
obtained from the finger or the ear often show a certain amount of 
hemolysis. Obtaining blood in quantity from the ear lobe will be facil- 
itated by making two punctures about 5 mm. apart on the lower 
portion of the ear lobe with a well sharpened triangular lancet such as 
the one recommended on page 18 (Fig. 2, a). The line of puncture 
should be transverse to the margin of the ear lobe and the lancet 
should be pushed up into the tissues of the lobe to the depth of 1 mm. 
Usually a small vessel will be cut by this technic and the blood flows 
fairly free into the test-tube. 

The method of choice is very distinctly the puncture of an arm vein, 
a procedure which is simplicity itself, under ordinary conditions and 



THE WASSERMANN REACTION 



123 



should not cause the patient any inconvenience or more than trivial 
pain if properly performed. It is absolutely essential that the needle 
be sharp. A fine oil stone and a bit of emery paper should be part of 
the regular equipment. Rust may be removed from the shaft of the 



A 




n\ 




Fig. 32. — Apparatus for immunology, a, Mohr pipette, 5 and 10 cc. capacities, 
graduated in 0.1 and 0.5 cc. respectively; 6, 1 cc. pipettes, graduated in 0.1 cc. and 0.01 
cc. ; c, glass-stoppered graduated mixing cylinders; d, author's device for obtaining 
blood; e, MacRae needle; /, Keidel tube; g, Keidel tube as modified to fit centrifuge 
shield; h, dental brooch for removing cotton plugs from pipettes; i, Blackfan apparatus 
for securing blood from children; j, Luer syringe; k, capillary pipette with mouth-piece 
and rubber connection. 



needle with the emery paper. The point should be carefully perfected 
by sharpening on the oil stone. Many needles come from the makers 
improperly ground so that behind the point is a high shoulder, as shown 
in Fig. 33. When this is found it is well to grind down the "hump" 



124 



EXAMINATION OF THE BLOOD 



on a carborundum stone until a proper point is secured. The care of the 
needle is also of great importance. It should be washed out with water 
immediately after use to remove the blood from its lumen, it should 
be boiled and dried by running through alcohol followed by ether, 
unless it is to be used again the same day. 



□n> 



Up> 



Fig. 33. — A, needle-point improperly sharpened; B, needle-point properly sharpened. 

Several forms of apparatus are available. A bare needle may be 
used. It comes with the outfits furnished by many of the large public 
laboratories. It will be found especially bothersome with small veins. 
It may be inserted with the hand or by gripping it with a stout pair of 
artery forceps (Fig. 34) . 




Fig. 34. — Thompson's apparatus for collecting blood. 

Partial vacuum is often desirable. The Macliae needle has the 
advantages of dependability and simplicity and a small number of 
parts to get out of order (Fig. 32, e). Here vacuum is obtained by 
removing the air from the tube by suction. A glass mouth-piece 
stuffed with cotton may be attached to the rubber tubing to obviate 
any possibility of drawing the patient's blood into the- operator's 
mouth, but this accident could not occur if rubber tubing is kept above 
the needle in the position shown in the sketch. 

Stiff has devised a piece of apparatus employing the same principle 
which can be improvised readily in the laboratory at trifling expense 
from glass and rubber tubing, a rubber stopper and an ordinary needle. 



THE WASSERMANN REACTION 



125 



The writer's device, in which an attempt is made to combine the best 
features of the MacRae and Stitt needles, is more convenient than 
Stitt's since it may be handled with one hand, and has the advantage 
over the MacRae needle that there are no metal parts to rust except 
the needle proper. The size of the needle may be selected for the 




c. s?s E. CO. 
Fig. 35. — Rack. 



particular case (Fig. 32, d and 37) - 1 A solid glass syringe of the Luer 
type (Fig. 32, j) with a slip-joint end to fit the needle, may be used. 
A syringe which necessitates a washer in the needle is an abomination. 
Cheap glass syringes prove to be an aggravation, since poor workman- 
ship may lead to leakage and insufficient vacuum. 




The Keidel tube is convenient. This consists of a sealed glass bulb 
from which the air has been exhausted, to which is attached, by means 
of rubber tubing, a needle fitted with a stylet. The needle and tubing 
are covered with a small test-tube (Fig. 32, /). When one desires 
to obtain a specimen of blood, the test-tube covering the needle is 



This is manufactured by the Will Corporation, Rochester, N. Y. 



126 EXAMINATION OF THE BLOOD 

removed, the stylet withdrawn, and the needle inserted through the 
sterilized skin into the vein. With a pair of hemostatic forceps, the 
glass tip of the vacuum bulb is crushed by pressure on the glass con- 
nection and blood should rush into the bulb. When filled it is sealed 
by replacing the stylet and covering the needle with the test-tube. 

The Keidel tube is especially convenient for those who desire to take 
a specimen of blood and send it to a laboratory. In the laboratory, 
however, they are nuisances, because it is difficult to remove the blood 
from them through the narrow neck, and centrifugalization to separate 
serum from clot before removal would be more or less out of the 
question since the size of the tube is not adapted to the centrifuge 
shields. The writer has attempted to obviate this difficulty by having 
tubes made of thick, tough glass and of the length and diameter to fit 
the Cornell centrifuge shield (Fig. 32, g). When they are received in 
the laboratory, the top may be removed and the tube put in the 
centrifuge to separate the serum. These tubes are made by the Steele 
Glass Co., Philadelphia, Pa. The chief objection to the Keidel tube is 
the cost, since they can be used only once. 

Technic of Venipuncture. — With any form of apparatus, the general 
procedure is the same. The patient is seated in a chair in such position 
that a good light falls on the arm. If the patient be in bed and the 
MacRae or the writer's needle be used, the arm should be so arranged 
that it will hang down over the edge of the bed. The sleeve is rolled 
up above the elbow and a piece of rubber tubing drawn tight around the 
arm above the elbow (Fig. 37). The tourniquet should not be tied, 
but should be drawn taut, and one end caught underneath as shown in 
the sketch so that the operator may readily release the tourniquet 
with one hand by pulling on this end. Experience will show that 
median cephalic and the median basilic veins exist only in text-books 
on anatomy and in rare individuals. The skin over the vein is cleansed, 
preferably by simply applying with a swab a mixture of equal parts of 
tincture of iodine and 95 per cent, alcohol. This should be allowed to 
dry. Distention of the vein may be accelerated by directing the 
patient to alternately clench and open the fist while the tourniquet is in 
position. When maximum distention has been secured, the needle is 
inserted through the skin over the vein in a direction parallel to its 
course and toward the patient's shoulder. Directing its insertion 
toward the hand is unnecessary and usually inconvenient (Fig. 37). 
A novice will find it easier to make a quick thrust to carry the point of 
the needle under the skin and a second one to push it into the lumen of 
the vein. If suction is employed, it may be used now; should blood 
fail to appear promptly the point of the needle should be withdrawn 
slightly, though not from underneath the skin, and reinserted until 
the wall of the vein has been penetrated. Care must be taken not to 
go through both anterior and posterior walls of the vein. W'hen the 
required amount of blood has been obtained, the tucked-under end of 
the rubber-tubing tourniquet may be pulled, thereby loosening it, 



THE WASSERMANN REACTION 



127 



the needle may be drawn out and a piece of gauze or cotton placed over 
the puncture. The patient is directed to make pressure on the cotton 
with his free hand for a few moments to arrest the bleeding and to 
prevent extravasation of blood from the vein into the tissues under the 
skin. Ordinarily the only dressing required is a drop of collodion. 




Fig. 37. — -Withdrawing blood with author's apparatus. Tourniquet is not knotted, 
but loop B is tucked under. Tourniquet may be detached by pulling on end A. 



Collection of Blood in Children.— The collection of blood in infants 
and children may offer some difficulties. (In such cases, the Noguchi 
reaction has the advantage of employing small amounts of all reagents, 
patient's serum included.) In infants, the cupping device of Blackfan 
is helpful (Fig. 32, i). The skin of the back below the scapula is scrub- 
bed with alcohol, and is scarified with a sharp scalpel or preferably, 
with a spring scarifier, previously sterilized by prolonged immersion 
in alcohol and then dried. The baby is held by an assistant, the 
cupping device is placed over the scarified area and a vacuum is 
created by the pump. Hyperemia of the cupped area results and blood 
flows fairly free. The attached test-tube should be held so that it is 
dependent in order that the blood may run into it. Usually 2 to 4 cc. 



12S EXAMINATION OF THE BLOOD 

may be obtained without especial difficulty. Ordinarily in older 
children, from two to four years of age and over, a satisfactory arm 
van may be found, but care should be taken to use a sharp needle of 
small bore and to have the child securely held. 

Whenever congenital syphilis is suspected, the blood should be 
obtained from the infant at the time of delivery by bleeding from the 
child's end of the umbilical cord before it is tied. This should be a 
routine measure in the obstetrical wards and hospitals. 

Precautions.— Certain precautions are necessary in obtaining speci- 
mens. Enquiry should be made as to the recent use of alcohol, since 
the work of Craig and Nichols showed that the ingestion of beer and 
whisky in considerable amounts has converted a strongly positive result 
into a negative one when tests were made within twenty-four hours 
after taking the alcohol. Bacterial contamination of the blood serum 
must be guarded against, for Craig found that growth in the serum of 
certain common bacteria such as staphylococci and streptococci may 
produce thermostabile anticomplementary substances which would 
seriously interfere with the reaction. Sera which are heavily charged 
with bile are often anticomplementary. It is also asserted that blood 
taken after ether or chloroform anesthesia occasionally shows a false 
positive result. (Kolmer.) 

The specimen tube should be labelled at once to prevent possible 
confusion, promptly stoppered with a clean cork, and placed in an 
upright position to clot. Stoppering with cotton or gauze is inadvis- 
able since either material would absorb the serum should the tube be 
tipped by accident. 

When the specimen is to be examined on the same day, no especial 
care is necessary regarding its preservation. If it is to be examined 
on the following day, the serum may be separated when convenient and 
the specimen kept in the refrigerator over night. When the specimen 
is shipped to a distant city, the tube should be carefully packed in a 
stout container, taking care to press down the cork securely. When 
whole blood is shipped, it should be secured and mailed to reach the 
laboratory at the time when the laboratory does its Wassermann reac- 
tions in order to prevent decomposition. Absolute asepsis is advisable, 
since Craig has shown that contamination is apt to give false results 
or at least interfere with the reaction through anticomplementary 
substances. A wise precaution is to send separated and inactivated 
serum with a notation as to the nature and date of the procedure. 

Separation and Inactivation of Serum.— The blood is allowed to coagu- 
late and the clot separated from the wall of the tube by loosening it 
with a stiff platinum wire, previously heated and cooled. In some 
laboratories, wooden applicators are employed for this purpose. This 
is not objectionable when the applicators are clean and are used only 
for one specimen. The tube is balanced on a scale against a tube 
containing a suitable amount of water and the two tubes are placed 
opposite each other in the centrifuge. Centrifugalization is carried 



THE WASSERMANN REACTION 129 

on until there is complete separation of the serum from the coagulum. 
The serum is withdrawn with a capilary pipette fitted with a mouth- 
piece and rubber connection (Fig. 32, k) and placed in a clean tube, 
properly labelled. If centrifugalized one day and separated on the 
following morning, it is usually possible to pour the serum from the 
clot and by employing x care, to keep it free from cells and hemolysis. 
This effects a considerable saving of pipettes. The serum is inactivated 
by immersing the tube in the water-bath to a depth equal to at least 
one-half its length, keeping at a temperature not below 55° C. and not 
above 58° C. for thirty minutes. Electrically heated baths are con- 
venient. If a large quantity of serum is to be inactivated, it should be 
divided between two or three tubes. When the reactions are to be set 
up, 1 cc. is placed in a clean tube and 4 cc. of physiological saline solu- 
tion are added to make a 1 : 5 dilution. 

Reagents.— 1. Salt Solution.— Physiological saline solution (0.85 or 
0.9 per cent,). 

2. Sheep Cells.— Suspension (5 per cent.) of sheep's red blood 
corpuscles. 

3. Hemolytic Amboceptor. — Serum of rabbit, immunized against sheep 
corpuscles. 

4. Complement.— Pooled serum of guinea-pig's blood. 

5. Antigen. — Cholesterolized (0.4 per cent.) alcoholic extract of 
human, beef or guinea-pig heart. 

Dilution of reagents is made conveniently in graduated glass stop- 
pered cylinders (Fig. 32, c). 

Physiological Saline Solution.— This should' be prepared from 
chemically pure sodium chloride and freshly distilled water. The 
salt must be weighed carefully. Too small an amount will give an 
hypotonic solution which hemolyzes red cells promptly. A markedly 
hypertonic solution will inhibit hemolysis. Since in spite of care 
mistakes are sometimes made, it is well to make up each batch of salt 
solution in advance of the time when it will be used, and to test it out 
carefully. When 2 cc. are added to 0.5 cc. of 5 per cent, red cell 
emulsion, hemolysis should not occur. 

It is advisable to sterilize the flasks of saline if they are to be kept 
on hand for more than a few days. The level of the fluid in the flask 
should be marked on the side with a file scratch, and sterilization 
accomplished either by boiling for five minutes or by treatment with 
15 pounds of steam pressure in the autoclave for fifteen minutes. In 
either case the saline should be cooled and the lost volume restored to 
the mark by sterilized distilled water. 

Sheep-cell Emulsion.— Sheep's blood may be obtained by keeping 
a sheep and by withdrawing blood from the jugular by means of a 
syringe when desired. The inconvenience incident to keeping an 
animal of this size in the laboratory and of bleeding it are objectionable 
to some workers. Added to this is the fact that in time an anemia is 
produced if any considerable quantity be withdrawn regularly, and 



130 EXAMINATION OF THE BLOOD 

consequently the cells become less resistant. If an abattoir be con- 
veniently located, it is usually possible to procure the services of an 
employee at small cost who will secure fresh blood from a healthy sheep 
and bring it to the laboratory. 

The blood may be transported in a Mason jar, partially filled with 
sterile physiological saline to which has been added 2 per cent, sodium 
citrate. In this 5 volumes or less of sheep blood may be received and 
gently shaken to ensure adequate mixing. Another method is to 
provide a stout glass bottle (8 or 16 oz. capacity) with a wide "salt- 
mouth" and a good stopper, into which have been placed about two 
dozen pieces of glass rod or other bits of glass. This outfit should be 
sterilized by dry heat. When the blood is obtained in this outfit, 
it should not fill the bottle completely and it should be shaken vigor- 
ously for ten to fifteen minutes to obtain complete defibrination. The 
butcher should be instructed not to take the first blood from the 
animal but to let this run away in order to secure blood as free from 
animal's wool and infective material as possible. 

The desired quantity is placed in centrifuge tube and centrifugal ized 
until the cells are packed at the bottom of the tubes, when the super- 
natant fluid consisting of serum and diluent is removed by pipette and 
discarded. The tubes are then filled with sterile physiological saline 
with which the corpuscular mass is thoroughly mixed and again 
centrifugalized. This supernatant saline is again removed by pipette 
or by pouring and the process repeated at least twice. If the reactions 
are to be performed the same day, the suspension may be prepared at 
once, but if they are to be done later the cells should be kept in the 
refrigerator undisturbed under the fluid of the last washing in corked 
tubes until needed. 

It is claimed that cells may be kept a long period of time even with 
only these precautions. With this the writer does not agree, unless, 
possibly, especial care is taken in procuring them. It is to be remem- 
bered that the cells are living, and that as soon as they die they become 
worthless, even though they be uninfected. Our experience has been 
that in cold weather the reactions should be done within forty-eight 
hours tif the time when the sheep was bled, and that in the hot summer 
months the reactions should be done on the following day. If kept 
longer, the cells are apt to turn purple and hemolyze very easily while 
standing for the final reading. 

When ready to prepare the suspension, the supernatant salt solu- 
tion is removed and packed cells are measured with a graduated 1 cc. 
pipette into the desired quantity of salt solution. It is apparent that 
this suspension is bound to be inaccurate since the density of the 
sediment will vary with the speed of the centrifuge and other factors. 
A constant standard suspension is obviously much to be desired. It 
is convenient to standardize with a Sahli hemoglobinometer. With 
the standard tube of the hemoglobinometer filled with a mixture 
adjusted to give a reading of 100 per cent, with normal male blood, 



THE WASSERMANN REACTION 131 

place ytt HC1 in the graduated tube to the 20 mark, add to it 0.5 cc. 
of the sheep corpuscle emulsion, mix, allow to stand one minute and 
then add distilled water to make the diluted fluid match the standard, 
as in performing the hemoglobin test on human blood. The reading 
should be 100 per cent. Cells or salt solution may be added to the 
sheep suspension as required to bring to standard. 

Occasionally sheep corpuscles are obtained which are either more or 
less resistant to the action of the other members of the hemolytic 
system than usual. It has been our experience to encounter the 
former phenomenon occasionally. If increased resistance be well 
marked, it is advisable to secure fresh blood; if not marked, the strength 
of other members of the system may be adjusted to compensate. 

Preparation of Hemolytic Amboceptor. — (Serum of rabbit immunized 
against sheep corpuscles.) A healthy, well grown rabbit should be 
selected. The sheep cells should be obtained with as great regard to 
sterility as possible. They are "washed" with sterile physiological 
saline at least four times, as directed for the preparation of sheep-cell 
suspension, and finally freed of saline solution. The cells should be 
warmed to approximately body temperature by placing the containing 
tube in water at 40° C. 

Intravenous Injections.— A small sterile glass syringe fitted to a well 
sharpened needle of fine gauge is charged with cell emulsion. An 
assistant may hold the animal across his lap stretching the body by 
drawing the forelegs forward with one hand and the hind legs back 
with the other, or the animal may be wrapped in a pillow-slip, leaving 
the head out (Fig. 38). The operator draws the ear down and injects 
the blood slowly into the marginal vein. 

A rabbit box is very convenient and renders an assistant unneces- 
sary. This can be made quite simply by putting a hole in the top of 
a box about 16 inches long by 6 wide and 7§ deep. The hole is for the 
rabbit's neck, the head being outside the box, the body inside. This 
top is made in two pieces to permit withdrawal of one part so that the 
rabbit may be placed in the box (Fig. 42) . 

The following schedule of injections may be followed: 

Washed undiluted sheep 
Injection. corpuscles. 

First 1 . cc. 

Second 1.5 " 

Third 2.0 " 

Fourth 2.0 " 

The time-interval should be three to five days between the injections. 
Although it may be lengthened to seven days there is some danger of 
producing anaphylactic shock. Exactness of dosage is not a rigid 
requirement. 

Intraperitoneal Injections. — If preferred, the injections may be given 
into the peritoneal cavity. This has no especial advantage except 
possibly that the technic of injection is easier. Larger doses of cells 



132 



EXAMINATION OF THE BLOOD 



must be given, however, and there is greater danger of infection and 
consequently a higher mortality. A stouter needle should be used 
than that prescribed for the intravenous method. The animal is held 
by the assistant with head down, as shown in the illustration (Fig. 41). 



Fig. 33 




Fig. 38.- 



Fig. 40 Fig. 41 

-Injecting cells into lateral ear vein of rabbit. Rabbit wrapped in pillow- 



Fig. 39. — Making incision into central ear vein. 

Fig. 40. — Collecting specimen of rabbit's blood for preliminary amboceptor titer. 

Fig. 41. — Injecting cells intraperitoneal^. 




Fig. 42. — Rabbit box, showing sliding top and hole for animal's head. 



The hair is washed with alcohol and the needle is inserted in the 
median line a little below a point midway between ensiform cartilage 
and the pubis. A small spot may be shaved, but this is really not 



THE WASSERMANN REACTION 133 

necessary, since the hair may be separated and " plastered down" with 
the alcohol. The cells should be warmed and injected slowly. Injec- 
tions may be given according to the following schedule. 

Washed sheep corpuscles 
Injection. diluted 50 per cent. 

First 5 cc. 

Second 10 " 

Third 10 " 

Fourth 10 " 

Untoward Symptoms.— In giving injections intravenously, untoward 
symptoms may develop if cells are injected so rapidly that the right 
heart is overloaded. With any mode of injection, anaphylactic symp- 
toms may appear, particularly with longer time intervals. It is worth 
while to attempt saving the rabbit by an intravenous injection of 1 
to 2 mm. adrenalin chloride (1 : 1000) solution. 

Preliminary Titer.— The titer of the serum may be determined at 
any time. It is the writer's practice to give 4 injections by the intra- 
venous route, then to wait seven days, secure blood and do a prelimi- 
nary titer. Almost invariably serum of high potency is obtained with 
3 or 4 injections. If the titer is low, an additional injection may be 
given. Blood is obtained easily by making a small incision with a 
small, sharp-pointed scalpel over the median vein of the ear, carrying 
the incision through the skin and across the vein, allowing 2 or 3 cc. 
of blood to run into a test-tube (Figs. 39 and 40). The animal may be 
held across an assistant's lap as for intravenous injections. Bleeding 
is stopped by making firm pressure over the incision for a few moments 
with a pledget of cotton. The blood in the tube is allowed to clot and 
is centrifugalized, when the serum is removed and inactivated at 56° C. 
for thirty minutes. It is now ready for titration. For titration 
complement should be used from pooled guinea-pig's serum which has 
been titrated against an already standardized hemolytic amboceptor. 
A 5 per cent, emulsion of sheep corpuscles is used, standardized as 
specified above. A row of 12 tubes is set up in a test-tube rack, placing 
a single tube in the rear row as a control (Table A). In the first tube 
is placed 0.8 cc. of physiological saline and in each of the remaining 
eleven in the front row is placed 0.5 cc. To the first tube is added 0.2 
cc. of the rabbit serum to be tested. For this purpose a 1 cc. pipette 
graduated in 0.01 cc. should be used. In the first tube we now have 
0.2 cc. of immune serum and 0.8 cc. of salt solution, the total being 
1.0 cc. The pipette is emptied, the mixture in the first tube is drawn 
into the pipette and blown back into the tube 2 or 3 times to ensure 
thorough mixing and finally 0.5 cc. is placed in tube No. 2. It is evident 
that we have left in tube No. 1 0.5 cc. of mixture and that there is 
present 0.1 cc. of immune serum (or one-half of the amount put in at 
first). We have carried into tube No. 2, 0.5 cc. of the mixture, which 
also contains 0.1 cc. of immune serum. The contents of tube No. 2 
are thoroughly mixed as before and 0.5 cc. of the mixture is put in tube 
No. 3. Now we have only 0.05 cc. of undiluted serum left in tube 



134 



EXAMINATION OF THE BLOOD 



No. 2. The process of mixing is repeated with tube No. 3 and its 
contents divided, and so on until tube No. 12 is reached. When tube 
No. 12 has received one-half the contents of tube No. 11, mixture is 
made and 0.5 cc. of the resultant mixture discarded. The quantities 
of undiluted immune serum are shown in the table. To each tube is 
now added 0.25 cc. of complement dilution (prepared so that this 
amount will contain one unit), 0.5 cc. of sheep corpuscle emulsion, and 
1.25 cc. of physiological salt solution. The control tube is set up by 
putting in 0.1 cc. of undiluted immune serum, 0.5 cc. of sheep corpuscle 
emulsion, and 1.9 cc. of normal salt solution. The rack is placed in the 
water-bath at 37° C. for one hour shaking the tubes every ten to 
fifteen minutes. The reading is then taken, that is, the last tube is 
noted in which complete hemolysis has taken place. The amount of 
immune serum in this tube represents one unit of hemolytic amboceptor. 
Of course there should be no hemolysis in the control tube. 

Ordinarily hemolysis should be complete at least in tube No. 8, that 
is, the serum should be so strong that about 0.0006 cc. or less will 
contain one unit, and it is our custom to secure sera of which 0.00006 
cc. to 0.0003 cc. or less will contain one unit. If the strength of the 
serum be sufficient, the animal may be bled to death. Of course this 
titration is crude, the results are only approximate and it is meant 
only to determine whether or not immunization has been successful. 
Such a titration is not accurate enough to permit its use in finally 
determining the dilution of the amboceptor previous to its actual use 
in the reaction. There should be no hemolysis in control tube No. 13 
because it contains no complement. In the hypothetical instance 
shown in Table A, one unit is 0.00019 cc, since this is the least amount 
which shows complete hemolysis. 

TABLE A. — PRELIMINARY TITER OF THE HEMOLYTIC AMBOCEPTOR. 





Actual amount 


Additional 

physiological 

saline, 

cc. 1 


Amount of 


Amount of 






Tube 
No. 


of undiluted 
immune rab- 


diluted 
complement 


0.5 per cent, 
sheep corpuscle 




Results in 
hypothetical 


bit serum, 


( = 1 unit), 


emulsion, 




instance. 1 




cc. 


cc. 


cc. 






1 . ' . . 


0.1 


1.25 


0.25 


0.5 




C.H. 


2 . 




0.05 


1.25 


0.25 


0.5 


u 
o 


C.H. 


3 . 




0.025 


1.25 


0.25 


0.5 




C.H. 


4 . 




0.0125 


1.25 


0.25 


0.5 


o 


C.H. 


5 . 




0.0062 


1.25 


0.25 


0.5 


m 


C.H. 


6 . 




0.0031 


1.25 


0.25 


0.5 


sg 


C.H. 


7 . 




0.00155 


1.25 


0.25 


0.5 


JS-° 


C.H. 


8 . 




0.00078 


1.25 


0.25 


0.5 




C.H. 


9 . 




0.00039 


1.25 


0.25 


0.5 




C.H. 


10 . 




0.00019 


1.25 


0.25 


0.5 


"3 


C.H. 


11 . 




0.000095 


1.25 


0.25 


0.5 


s 


P.H. 


12 . 




0.000042 


1.25 


0.25 


0.5 


a 


N.H. 


13 . 




0.1 


1.9 





0.5 




N.H. 


1 TV. 










• 


fn-~ 4- 





This amount is in addition to that originally placed in the tubes for the purpose of 
fractionating the immune serum. 

8 C.H. means complete hemolysis; P.H., partial hemolysis; and N.H., no hemolysis. 



THE WASSERMANN REACTION 135 

Bleeding the Babbit.— First Method.— The anterior surface of the 
rabbit's neck and the upper part of the chest is lathered with shaving 
soap, shaved and washed with alcohol and ether. A gauze pad soaked 
with alcohol is held in place over this area by an assistant. The 
fore legs are tied together with a stout cord, one end of which should 
be passed to a second assistant who is to hold the animal. The hind 
legs are also tied together. In this way one assistant is able to hold 
back both pairs of legs. The animal is now turned belly down, the 
first assistant holding the head out on a stretch and etherizing the 
animal, the second assistant aiding by pulling the fore and hind legs 
posteriorly and steadying the body. When etherized, the animal's 
neck is held over a wide sterile funnel inserted into a sterile 50 cc. 
centrifuge tube. The alcohol-soaked pad is removed, the neck dried 
with sterile gauze and the vessels of the neck cut with a quick 
sweep of a sharp amputating or autopsy knife, previously sterilized 
by prolonged immersion in 95 per cent, alcohol and dried with sterile 
gauze. A second sterile 50 cc. centrifuge tube should be standing in 
readiness in the rack, since a little over 50 cc. of blood are usually 
secured. When the animal is bled out, the tubes of blood are corked 
with sterile corks and placed in the refrigerator. 

Second Method— The rabbit is tied down on a board, etherized, and 
a long incision made through the skin, dissecting out the carotid 
artery under sterile conditions. The artery is tied off and nicked but 
not cut entirely across on the heart side of the ligature. The blood is 
allowed to spurt into a sterile wide mouth tube. Some workers insert 
a fine cannula into the artery. While this method is theoretically 
irreproachable on grounds of asepsis, longer etherization is required 
and the mortality is higher. It is somewhat more difficult technically 
and etherization reduces the titer of the amboceptor. 

Third Method.— The animal is tied down on a rabbit board. The 
hair is plucked from the inguinal region, the skin sterilized by applying 
alcohol, and a long incision made through it over and parallel to 
Poupart's ligament. The edges of the incision are retracted and a cut 
made through the fascia, which is carefully separated. This is an 
important step in the procedure. Then the femoral vessels are cut with 
the knife and the blood is allowed to flow into a sterile 50 cc. centrifuge 
tube or Erlenmeyer flask, pressing the mouth against the exposed 
tissues close to the bleeding vessels. Etherization is unnecessary and 
the operation may be performed quickly. The only instruments 
required are sterile knife and dissecting forceps. 

Preparation and Preservation of Hemolytic Amboceptor.— It is prefer- 
able to allow the blood to stand in the refrigerator over night since 
the serum gains slightly in potency. On the following day it may 
be separated exactly as in the manner described in the preparation 
of human" blood with a sterile capillary pipette. The bulk of the 
serum is divided among 3 or 4 sterile test-tubes (size f x 6 inches), while 
into each of 7 or 8 small tubes (size f x 4 inches) are placed 0.5 cc. 



136 



EXAMINATION OF THE BLOOD 



The latter amounts must be measured out with a 1 cc. pipette, gradu- 
ated in 0.1 cc. All of the tubes are sealed in a blowpipe or the hot 
flame of a Tyrill burner. The smaller tubes are for use in the pre- 
liminary titrations. As the need arises the larger tubes may be 
opened and divided up into small tubes containing 0.5 cc. each. It is 
wise to keep all tubes in the ice-box. In some laboratories it is the 
custom to keep the serum in bulk in a bottle from which serum is 
pipetted whenever needed. This incurs the not inconsiderable risk of 
contaminating a valuable reagent. 

When amboceptor is desired, one of the small sealed tubes containing 
0.5 cc. is opened at the top by nicking with a sharp file, removing the 
upper portion with a sharp blow. Into the tube is placed 4.5 cc. of 
physiological salt solution, which gives an ample quantity of 1:10 
dilution for titration and the day's reactions. Immune hemolytic 
serum may be preserved in this manner for many months with no 
change in the titer. 

TABLE B. — TABLE SHOWING TITER OF HEMOLYTIC AMBOCEPOTR. 









Amount of 
0.9 per ct. 
NaCl solu- 
tion, 


Amount of 


Amount of 






Actual 


Tube 
No. 


Ambocep- 
tor in dilu- 
tion to be 


Amount of 
dilution. 


diluted 
comple- 
ment ( = 1 


5 per cent, 
sheep cor- 
puscle 




Result in 
hypotheti- 
cal case. 


amount of 

undiluted 

ambo- 




used. 


cc. 


unit), 


emulsion, 




ceptor, 










cc. 


cc. 






cc. 


1 . . 


1:10 


0.1 


1.65 


0.25 


0.5 




C.H.i 


0.01 


2 . . 


1:100 


0.1 


1.65 


0.25 


0.5 


o 


C.H. 


0.001 


3 . . 


1:1000 


0.5 


1.25 


•0.25 


0.5 


O 


C.H. 


0.0005 


4 . . 


1:1000 


0.4 


1.35 


0.25 


0.5 




C.H. 


0.0004 


5 . . 


1:1000 


0.3 


1.45 


0.25 


0.5 


*!* 


C.H. 


0.0003 


6 . . 


1:1000 


0.25 


1.50 


0.25 


0.5 


S3 o 


C.H. 


0.00025 


7 . 


1:1000 


0.2 


1.55 


0.25 


0.5 


■g <D 


C.H. 


0.0002 


8 . . 


1:1000 


0.15 


1.60 


0.25 


0.5 


fo 


C.H. 


0.00015 


9 . . 


1:1000 


0.1 


1 . 65 


0.25 


0.5 


+J 


M.H. 


0.0001 


10 . . 


1:10000 


0.9 


0.85 


0.25 


0.5 


is 


M.H. 


0.00009 


11 . . 


i: ioooo 


0.8 


0.95 


0.25 


0.5 




P.H. 


0.00008 


12 . . 


1 : 1 2 


0.1 


1.90 


° 


0.5 




N.H. 


0.01 



Final Determinations of the Hemolytic Amboceptor Titer.— Before 
finally deciding upon the final titer of a new amboceptor, it should be 
titrated on several occasions, using complement from pooled guinea- 
pig sera the unit of which has been determined by titration against a 
tried amboceptor. Having prepared a 1:10 dilution of amboceptor 
as directed in the preceding section, a 1 : 100 dilution should be made 
from this by mixing 1 cc. of the 1 : 10 dilution with 9 cc. of physiological 
saline. From this in turn is made a 1 : 1000 dilution by taking 1 cc. 
(of the 1 :100 dilution) and adding to 9 cc. of saline. Likewise from 

1 C.H. means complete hemolysis; M.H., marked hemolysis; P.H., partial hemolysis; 
N.H., no hemolysis. 

2 Tube No. 12 is the control and should show no hemolysis. 



THE WASSERMANN REACTION 137 

this is made a 1 : 10,000 dilution by taking 1 cc. (of the 1 : 1000 dilution) 
and adding the 9 cc. of saline. We will assume that the preliminary 
titer showed that one unit was equal to about 0.00019 cc. A rack of 
tubes would be set up as indicated in Table B, putting into each the 
amount of diluted hemolytic serum specified of the dilution specified 
and adding 0.25 cc. of diluted complement (containing one unit of 
complement), 0.5 cc. of sheep-cell emulsion, and physiological saline 
solution sufficient to bring the total volume in each tube to 2.5 cc. 

According to the table, it is seen that one unit of amboceptor, that is, 
the least quantity which will hemoly&e the sheep corpuscles in the presence 
of one unit of complement, is equal to 0.00015 cc. The titration is 
repeated on three or four occasions and the average of the results 
taken. When the amboceptor is finally used in performing the reac- 
tion, it must be diluted so that 0.5 cc. will contain 2 units. The follow- 
ing equation will be of assistance in determining the amount of saline 
solution to be added to a given quantity of 1 :10 dilution of hemolytic 
amboceptor: 

X represents the total quantity of diluted amboceptor. 
A represents the amount of undiluted amboceptor which is used. 
0. 5 is the amount of the diluted amboceptor which we desire to contain 2 units. 
B represents the quantity of undiluted amboceptor equivalent to 1 unit. 

X : A =0.5 : 2B. 

Example.— Suppose that we desire to start with 1 cc. of our 1 :10 
dilution of amboceptor (or 0.1 cc. of undiluted amboceptor) and that 
the titer shows 1 unit equals 0.00015 cc, as illustrated above: 

X : 0.1 = 0.5 : 2(.00015) 
X = 166.7 

That is, 1 cc. of 1 : 10 dilution (equivalent to 0.1 cc. of undiluted 
amboceptor) is to be diluted up to a total volume of 166.7 cc. with 
physiological salt solution. In other words, we desire a dilution of 
1 to 166.7. We may check the correctness of our calculation in this 
way. With 166.7 cc. of diluted amboceptor, we have 333.4 doses of 
diluted amboceptor 0.5 cc. each. Each dose, therefore, would contain 
g-jrg- of undiluted amboceptor (0.1 cc), or .0003 cc, which is what is 
desired. 

In titrating complement titer before each day's work, it is advisable 
to repeat the titer as a check on the indicator system for while the 
amboceptor itself is almost stable, such a titer serves as an excellent 
supplement to the complement titer. If desired, several of the tubes 
may be omitted, leaving those which are near the hemolytic unit. 

Preparation and Titration of the Complement. — Complement is 
obtained from guinea-pig blood. The use of pooled serum from three 
or four pigs weighing 400 gm. or more is advisable since specimens 
from different pigs vary considerably. In order to prevent dilution 
of the blood by hydremia, the pigs may be fed on an exclusive diet of 
oats for at least a day before they are bled. It is poor economy to 



138 



EXAMIXATIOX OF THE BLOOD 



use small pigs, since the yield of blood is scanty and weak in comple- 
ment. 

Serum may be procured either by cutting the animals' throats and 
bleeding them to death, or by aspirating blood with a syringe from the 
right auricle. The latter method has the advantage of economy, 
since the pig may be kept alive for some time to furnish many small 
lots of complement, but an anemia is produced resulting in weak 
complement. 





Fig. 43. — Etherizing guinea-pig. 



Fig. 44. — Stunning pig with sharp blo\ 
in occipital region. 





Fig. 45. — Cutting throat. 



Fig. 46. — Bleeding through a funnel 
into test-tube. 



Methods for Obtaining Blood.— (a) The animals may be bled into 
Petri dishes, but preferably through a funnel into test-tubes, since in 
the latter way centrifugalization and separation of the serum is facil- 
itated. In a test-tube rack are placed several tubes which will fit the 
centrifuge and into one is placed a wide funnel. The animal may be 



THE WASSERMANN REACTION 139 

etherized lightly (Fig. 43), or stunned by a sharp blow at the base of 
the skull with the ulnar side of the hand (Fig. 44). An assistant 
draws the animal's head forward, the operator holds the fore-paws 
out of the way and pulls the body back, and severs the vessels of the 
neck with a long knife (Fig. 45) while the neck is held Over the funnel 
(Fig. 46). (b) A needle about 1 inch long of 18 gauge is attached to a 
glass syringe (Fig. 32, j) of Luer type. The pig is etherized lightly and 
the hair clipped off in the precordial area. It is held firmly, preferably 
by an assistant. The point of maximum cardiac pulsation is selected, 
the skin sterilized with iodine, and the needle inserted between the ribs 
at this point, withdrawing about 5 cc. of blood (Fig. 47). 




Fig. 47. — Method of bleeding guinea-pig for complement. (Thompson.) 

Separation and Care of Serum.— When possible, the serum is allowed 
to stand on the clot for several hours, preferably over night. It may 
be separated in the manner directed for human blood serum (page 128). 
The serum for all tubes should be mixed, preferably in a glass-stoppered 
graduated cylinder. 

Especial care must be taken to keep the complement cold at all 
times except when in actual use. Both before and after separation 
and again after final dilution, it should be placed in the refrigerator. 
It should not be shaken, but may be mixed by inverting the glass 
cylinder. The worker should remember that complement is unlike 
amboceptor in that it is thermolabile and readily deteriorates at higher 
temperature. 

Titration of Complement.— In titrating the complement, amboceptor 
of known strength should be used and diluted so that 0.25 cc. of the 
dilution contains 1 unit. A 5 per cent, sheep corpuscle emulsion 
should be prepared and standardized as directed (page 129) and a 1 : 10 
dilution of pooled complement prepared by adding 9 cc. of physio- 
logical saline to 1 cc. of serum. The titer is set up as indicated in 



140 



EXAMINATION OF THE BLOOD 



Table C, placing graduated amounts of complement dilution in the 
tubes, each receiving 0.25 cc. hemolytic amboceptor dilution, 0.5 cc 
sheep corpuscle emulsion, and sufficient physiological saline to bring 
the total volume to 2.5 cc. Three controls are necessary, one con- 
taining amboceptor, corpuscles and saline only, to determine that the 
amboceptor does not produce hemolysis without complement; one with 
complement, corpuscles and saline only, to find out whether or not the 
complement itself contains a hemolytic substance; and another with 
corpuscles and saline only, to make certain that the cells do not hemo- 
lyze spontaneously. 

TABLE C— TABLE SHOWING COMPLEMENT TITER. 





Amount of 


Amount of 


Amount of 


Amount of 
0.9 per cent. 

NaCl solu- 
tion, 








Tube 

No. 


1 : 10 com- 
plement . 
dilution, 
cc. 


amboceptor 

in dilution 

(0.25 cc. = 

1 unit), 


5 per cent, 
sheep cor- 
puscle 
emulsion, 




Results in 
hypotheti- 
cal case. 


amount of 
undiluted 
complement, 






cc. 


cc. 










1 . . . 


0.5 


0.25 


0.5 


1.25 


6 


C.H.i 


0.05 


2 . . . 


0.4 


0.25 


0.5 


1.35 


•_ 


C.H. 


0.04 


3 . . . 


0.3 


0.25 


0.5 


1.45 


w fcl 


C.H. 


0.03 


4 . . . 


0.25 


0.25 


0.5 


1.50 


03 O 


C.H. 


0.025 


5 . . . 


0.2 


0.25 


0.5 


1.55 


'i s 


P.H. 


0.02 


6 . . . 


0.1 


0.25 


0.5 


1.65 


■9° 


S.H. 


0.01 


Control 1 





0.25 


0.5 


1.75 


«o 


N.H. 


0.0 


Control 2 


0.5 





• 0.5 


1.50 


t 


N.H. 


0.05 


Control 3 








0.5 


2.00 


a 


N.H. 


0.0 



According to the illustrative table, it will be seen that one unit of 
complement, that is, the least quantity which is sufficient to cause 
complete hemolysis of a unit of sheep cells in the presence of an excess 
of hemolytic amboceptor is 0.25 cc. of 1:10 dilution, or 0.025 cc. of 
undiluted complement. 

If the titer be set up with 2 units of amboceptor in each tube, the 
results when read at the end of ten minutes' incubation at 37° C. will 
be very similar to those obtained with the use of 1 unit in each tube, 
read at the end of sixty minutes. 

It is the author's practice to take advantage of this fact, setting 
up a "short" titer with 2 units of amboceptor in each tube, and obtain- 
ing a preliminary result in ten minutes. With this reading as an 
indication of the final result, a second titer is prepared using one unit 
of amboceptor in each tube. The accuracy of the determination is 
enhanced by employing a 1 : 20 dilution of complement instead of the 
customary 1:10 dilution. If, for example, the "short" titer showed 
one unit of complement to be approximately 0.2 cc, the final titer 
would be set up with 0.46 cc, 0.44 cc, 0.42 cc, 0.40 cc, 0.38 cc, 0.36 
cc, 0.34 cc, 0.32 cc. of 1:20 complement dilution, corresponding 

1 C.H., means complete hemolysis; P.H., partial hemolysis; S.H., slight hemolysis; 
N.H., no hemolysis. 



THE WASSERMANN REACTION 141 

respectively to 0.23 cc, 0.22 cci, 0.21 cc, 0.19 cc., 0.18 cc, 0.17 cc, 
0.16 cc. of 1:10 dilution. 

Interpretation of the Results.— A complement should not be employed 
which is so weak that it will not produce complete hemolysis in tube 
No. 2 (that is, with 0.04 cc. undiluted complement), or which is so 
strong that it produces complete or nearly complete hemolysis in tube 
No. 6 (that is, with only 0.01 cc. undiluted complement). A comple- 
ment is not to be employed which shows hemolysis in control tube No. 2. 

This titer having been completed, a small amount of complement is 
diluted accordingly, and a set of trial reactions is prepared. These 
serve as a check on the balance between the members of the hemolytic 
system, as will be explained later (for number of units to be employed, 
see page 147) . 

In diluting the complement, the following equation may be helpful: 

X represents the total quantity of diluted complement. 
A represents the amount of undiluted pooled serum to be used. 
0.5 cc. is the amount of complement dilution which we desire to contain 1.5 units. 
B represents the quantity of complement found on titration to be 1 unit. 

Example.— 

X : A = 0.5 : 1.5B. 

Now suppose that we wish to dilute 8 cc. of guinea-pig serum, and 
that the titer shows (as in above table) that 1 unit =0.025 cc. 

X : 8 = 0.5 : 1.5 (0.025) 

X = 106.6 

Then 8 cc. of serum are diluted by adding 98.6 cc. of physiological 
salt solution to make the total volume 106.6 cc. 

The Antigens.— Preparation. — Cholesterolized Alcoholic Extracts of 
Normal Organs. —Human, beef, or guinea-pig hearts may be used. 
Human heart should be obtained preferably from an accident case 
or at least one free from infection, and as soon after death as possible. 
Guinea-pig hearts may be removed from animals which have been 
bled out to secure complement, prepared as directed in the following 
paragraph and placed with a suitable amount of alcohol in a bottle. 
Additional hearts with proportional amounts of alcohol may be added 
from time to time. 

The heart-tissue should be freed from blood, using only myocardium 
and removing fibrous and fatty tissue. The desired amount is weighed, 
cut into small pieces, and ground in a clean dry mortar with bits of 
clean broken glass. The resulting paste-like mixture is taken up with 
a spoon and put into a clean Erlenmeyer flask or Mason jar. Absolute 
alcohol should be added in the proportion of 100 cc. of alcohol to each 
10 gm. of heart. The receptacle should be tightly stoppered and kept 
in the incubator at 37° C. about seven days when it is cooled by placing 
in the refrigerator over night. After cooling, the extract is filtered 
through fat-free filter paper and the filtrate kept in a dark bottle in the 
refrigerator. To 100 cc. is added 0.4 gm. cholesterol (C. P.). This is 



142 EXAMINATION OF THE BLOOD 

dissolved in the antigen by placing it in the incubator at 37° C. over 
night when the solution is cooled at room temperature and filtered, if 
any sediment be seen. Cholesterolized antigens are of remarkably 
uniform strength. 

Alcoholic Extract of Syphilitic Liver.— A liver from a syphilitic fetus 
is obtained in as fresh a condition as possible and washed free from 
blood. A portion is weighed and ground in a mortar with a pestle 
with pieces of broken glass. The resulting mass is placed in a Mason 
jar or a wide mouthed flask or bottle and to it is added absolute alcohol 
in the proportion of 100 cc. of alcohol to each 10 gm. of liver tissue. 
The mixture is placed in the incubator from seven to ten days, shaking 
well each day, when it is filtered through filter paper. The filtrate 
should be titrated. If the antigenic strength proves insufficient, the 
filtrate may be evaporated to two-thirds its original volume by placing 
it in an open dish in front of an electric fan. (Craig.) 

Nogvchi Acetone— Insoluble Antigen.— Normal fat-free heart-muscle 
is ground up and absolute alcohol added (100 cc. alcohol to 10 gm. 
tissue). This is kept at 37° C. for seven days, shaking each day, and 
then filtered. The filtrate is evaporated to dryness with an electric 
fan. This step ordinarily requires fifteen to twenty-four hours. The 
residuum is dissolved in ether and the opalescent ethereal extract 
allowed to stand in the refrigerator over night so that the sediment may 
settle. In the morning, the clear supernatant fluid is poured off and 
evaporated in front of an electric fan to small bulk. Care should be 
taken to avoid igniting the ether. Approximately 10 volumes of chemic- 
ally pure acetone are added to the residuum. The acetone mixture is 
put aside for several hours and the acetone decanted. In this way 
cholesterol is discarded, since it is acetone-soluble; and the sticky 
brown precipitate, made up of lecithin and certain acetone-insoluble 
lipoids, is collected with a spatula and kept in a bottle under acetone. 
To prepare for use, about 0.3 gm. of the acetone-insoluble precipitate 
is dissolved in 1 cc. of ether and 9 cc. of methyl alcohol are added. 
This may be kept as stock solution and a small quantity diluted 1 : 10 
with physiological saline as required for the test. 

Rapid Method of Preparing Antigens from Normal Heart Muscle.— 
Ecker and Sasano adopt the suggestions of Erlandsen and of Neymann 
and Gager to extract finely divided heart muscle, dried in air, with 
ether and then alcohol, discarding the ethereal extract which contains 
no substances of antigenic value except small quantities of lecithin 
which has high anticomplementary power. Normal heart tissue is 
freed from fat, larger bloodvessels, endocardium and pericardium, 
ground finely, spread on glass plates, and dried at incubator tempera- 
ture or in front of an electric fan. The dried material is powdered in 
a mortar. This may be kept in bottles for some time. Twenty-five 
gm. are extracted 3 times, using 50 cc. of ether each time for a period of 
ten minutes. The ether is discarded and the heart tissue allowed to 
dry, when it is mixed with 75 cc. of 95 per cent, alcohol in a flask and 



THE WASSERMANN REACTION 



143 



extracted in a reflux condenser over a boiling water-bath for three 
hours. A reflux condenser may be made by inserting a 40-inch glass 
tube of 3 mm. diameter through the stopper of the flask, supporting 
this in a vertical position so that the condensed alcohol will run 
back into the flask. An antigen prepared in this way may be titrated 
in dilutions of 1:10, 1:50, 1:100, 1:150, 1:200, using 0.5 cc. of each 
dilution with known positive serum, and twice the quantity of each 
dilution with negative serum. Ecker and Sasano found that it was 
usually possible to employ these antigens in dilutions of 1 :20. 

Titration of the Antigen.— The following properties should be 
determined by titration: (1) hemolytic action; (2) anticomplementary 
action; (3) specific binding power. The balance of the hemolytic 
system must be properly adjusted and positive and negative sera must 
be on hand. The positive serum must be titrated as shown in Table D. 
A standardized antigen is desirable. The method of setting up the 
necessary titers is shown in Parts I to V of Table E. 

TABLE D.— DETERMINATION OF SYPHILITIC UNIT OF POSITIVE 
SERUM PRELIMINARY TO TITRATION OF ANTIGEN. 



Tube 
No. 


Actual 
amount 
of syphil- 
itic serum, 


Amount 
of stand- 
ard anti- 
gen, 
cc. 1 


Addi- 
tional 0.9 
per cent. 

NaCl, 
cc. 2 


Amount 
of diluted 

comple- 
ment ( = 
1.5 units), 
cc. 




Amount 
of diluted 
ambocep- 
tor ( = 2 
units), 
cc. 


Amount 
of sheep 
cell sus- 
pension 
(5 per ct.), 
cc. 




Results in 
hypotheti- 
cal case. 


1 . . 


0.1 


0.5 





0.5 




0.5 


0.5 




+ + + + 


2 






0.05 


0.5 





0.5 


_ o 


0.5 


0.5 


*J3 


+ + + + 


3 






0.025 


0.5 





0.5 


03 g 


0.5 


0.5 


N§ 


+ + + + 


4 






0.012 


0.5 





0.5 




0.5 


0.5 


^ 


+ + 


5 






0.006 


0.5 





0.5 


"g ° 


0.5 


0.5 




± 


6 






0.003 


0.5 





0.5 


*o 


0.5 


0..5 


PQ 


— 


7 






0.2 





0.8 


0.5 


1-1 co 


0.5 


0.5 


co 


— 



TABLE E, PART I.— DETERMINATION OF HEMOLYTIC QUALITIES. 





Amount of 


Amount of 


/ mount of 


Amount of 






Tube 


antigen dilution 


known negative 


sheep-cell 


0.9 per cent. 




hypothetical 


No. 


(1 = 10), 


serum, 


suspension, 


NaCl solution, 






cc. 


cc. 


5 per cent. cc. 


cc. 






1 . 


0.5 


0.1 


0.5 


1.4 




N.H.s 


2 . 


1.0 


0.1 


0.5 


1.9 


C30 El 


N.H. 



1 A known antigen, used and clinically tested, diluted according to previous titration, 
usually 1:10. 

2 This is amount in addition to that originally placed in the tubes for the purpose of 
fractionating the immune serum. 

3 N. H. means no hemolysis. 



144 



EXAMINATION OF THE BLOOD 



TABLE E, PART II. 



■DETERMINATION OF ANTICOMPLEMENTARY 
UNIT. 



Tube 
No. 


Amount of 

antigen 

dilution 

(1=10), 

cc. 


Amount of 
0.9 per ct. 
NaCl solu- 
tion, 


Amount of 

comple- 
ment dilu- 
tion (=1.5 
units), 
cc. 




Amount of 
ambocep- 
tor dilu- 
tion ( = 2 
units), 


Amount of 
sheep cell 
suspension, 
5 per cent., 
cc. 




Results in 
hypotheti- 
cal case. 


3 




0.5 


0.5 


0.5 


?- 


0.5 


0.5 




C.H. 1 


4 




1.0 





0.5 


58 O 


0.5 


0.5 


eg o 


C.H. 






1.25 





0.5 


-~ 


0.5 


0.5 


«■ a 


; C.H. 


fi 




1.5 





0.5 


•*? ° 


0.5 


0.5 




C.H. 


7 




2.0 





0.5 


| 


0.5 


0.5 


B^ 


P.H. 
or N.H. 



TABLE E, PART III.— DETERMINATION OF ANTIGENIC UNIT. 



Tube 
No. 



Amount 
of anti- 
gen dilu- 
tion, 
cc. 



Amount Amount Amount 
of diluted of 0.9 per of com- 
syphilitic cent. Na- plement 
serum (= CI solu- ] dilution 

1 unit), tion, ( = 1.5 u.), 



8 


0.05 


0.1 


0.9 


0.5 


« ►: 


9 


0.10 


0.1 


0.8 


0.5 


a o 


10 


0.20 


0.1 


0.7 


0.5 




11 


0.30 


0.1 


0.6 


0.5 




12 


0.40 


0.1 


0.5 


0.5 


a" 5 


13 


0.50 


0.1 


0.4 


0.5 


Jj° 





Amount 






of ambo- 


of sheep 




Results 


ceptor 


cell sus- 




in hypo- 


dilution 


pension 




thetical 


(= 2 un.), (5perct.), 




case. 


cc. 


cc. 






0.5 


0.5 


r- 


C.H.* 


0.5 
0.5 


0.5 

0.5 


■53 § 


P.H. 

X.H. 


0.5 


0.5 




N.H. 


0.5 


0.5 




N.H. 


0.5 


0.5 


a^ 


X.H. 



TABLE E, PART IV.— DEMONSTRATION THAT ANTIGEN IS SPECIFIC. 



Amount Amount 
Amount Amount of 0.9 per of corn- 
Tube of antigen of normal cent. N a- plement 
No. dilution, serum, CI solu- dilution 
cc. cc. tion, ( = 1.5u.), 



cc. 



cc. 



Amount Amount 

of ambo- of sheep ! Results in 

ceptor cell sus- hypothet- 

dilution pension . ical case. 

(= 2un.),(5perct.), 



0.2 


0.1 


0.7 


0.5 


0.4 


0.1 


0.5 


0.5 


0.5 


0.1 


0.4 


0.5 



0.5 


0.5 


H 2 


0.5 


0.5 


C3 


0.5 


0.5 


%^ 



C.H.3 
C.H. 
C.H. 



N.H. means no hemolysis. 

N.H. means no hemolysis; P.H., partial hemolysis; and C.H., complete hemolysis. 

The standard antigen should be diluted so that 0.5 cc. will contain the required dose. 



THE WASSERMANN REACTION 



145 



TABLE E, PART V. — SERUM CONTROLS. 



Tube 
No. 


Amount 
of old 
stand- 
ardized 
dilution, 
cc. 1 


Amount 
of dilut- 
ed syph- 
ilitic 
serum 

{= } 
unit) , 

cc. 2 


Amount 

of 
normal 
serum, 

cc. 


Amount 
of 0.9 

per cent. 
NaCl 

solution, 
cc. 


Amount 
of com- 
plement 
dilution 
(=1.5 
units) , 
cc. 




Amount 
of ambo- 
ceptor 
dilution 
(=2 
units), 
cc. 


Amount 
of sheep 
cell sus- 
pension 
(5 p.c), 
cc. 




Results 
in hypo- 
thetical 
case. 3 


17 . . 

18 . . 

19 . . 

20 . . 


0.5 


0.5 




0.1 
0.2 









0.1 
0.2 


0.4 
0.8 
0.4 
0.8 


0.5 
0.5 
0.5 
0.5 


d 

il 

Is 


0.5 
0.5 
0.5 
0.5 


0.5 
0.5 
0.5 
0.5 


d 

JJ 

-S g 
1*" 


N.H. 
C.H. 
C.H. 
C.H. 



Determination of the Unit of Syphilitic Serum —For this a series of 
7 tubes are set up (see Table D). A serum should be selected which 
has given previously a strongly positive result. If it has been pre- 
served for more than a day or so, it should be inactivated again 
for fifteen minutes. It is desirable to select a serum which does not 
contain native antisheep amboceptor. In the first tube (Table D) is 
placed 0.2 cc. of syphilitic serum and 0.8 cc. of physiological saline. 
In each of the next five are placed 0.5 cc. of physiological saline. The 
contents of the first tube are mixed thoroughly, and 0.5 cc. of the mix- 
ture is transferred to the second tube. The resulting mixture is drawn 
up into the pipette and ejected back into the tube several times to 
ensure thorough mixing, and 0.5 cc. is removed and put into the third 
tube. The process is repeated until the contents of six tubes are mixed, 
when 0.5 cc. are discarded. We now have six tubes containing res- 
pectively 0.1 cc, 0.05 cc, 0.025 cc, 0.0125 cc, 0.006 cc, and 0.003 cc, 
of syphilitic serum.* The seventh tube is placed in the rear row of the 
rack and receives 0.2 cc, of undiluted syphilitic serum, 0.8 cc, of 
physiological saline and no antigen. Into each of the six tubes in the 
front row is placed the usual dose of the standard antigen. Into all 
seven tubes are placed 1.5 units of complement. The tubes are sub- 
jected to 37° C. for one hour and then 2 units of amboceptor and the 
usual amount (1 unit) of sheep cell suspension are added. They are 
again heated at 37° C. for one hour, when the results may be read. 
The least amount of syphilitic serum which causes complete inhibition 
of hemolysis (gives a + + + + reaction) is said to contain one syphilitic 
unit. In the hypothetical results given in the table, this occurs in 
tube No. 3, meaning that 0.025 cc. is one syphilitic unit. The serum 
should be diluted so that this amount will be contained in 0.1 cc, 



1 This is the same serum as that used in Part III, diluted in the same manner. 

2 This is the same serum as that used in Part IV. 

3 N.H. means no hemolysis; C.H. means complete hemolysis. 

10 



146 EXAMINATION OF THE BLOOD 

that is, to one part of serum is added three parts of physiological saline 
and 0.1 cc. of the mixture is used in titer. 

Dilution of Antigen for Titration.— A 1:10 dilution should be made 
of cholesterolized antigen, and a 1 :5 dilution of alcoholic organ extracts. 

Discussion of Titration.— If the antigen has no hemolytic action in 
the quantities used in tubes 1 and 2 of Part I of the titration, it may be 
regarded as acceptable on this score. The anticomplementary unit 
shown in the hypothetical instance, Part II of the titration, Tube No. 
7, is 2.0 cc. The dose of the diluted antigen to be used in the reaction 
may be that one-third of the amount (1.5 cc.) employed in tube No. 6, 
or one-third of the greatest amount which had no anticomplementary 
effect. This amount would be 0.5 cc. of a 1 : 10 dilution. That it is 
satisfactory from the standpoint of antigenic power is shown in Tube 
No. 10 of Part III of the titration, since so small an amount as 0.2 cc. 
of the 1 : 10 dilution causes complete fixation of the complement in the 
presence of one syphilitic unit. (The amount required here for com- 
plement fixation is considerably greater than would be needed if the 
syphilitic unit of the positive serum had not been determined and if 
the positive serum from an active case containing a number of syphilitic 
units had been employed. Had 0.1 cc. of serum of this sort been uti- 
lized in the antigen titer without titration of the syphilitic unit and 
without corresponding dilution, it would have a high binding-power 
and a new antigen w T ould have shown complete fixation of the comple- 
ment in much smaller amounts in the titer. It is evident that unless 
the syphilitic unit be ascertained, there will be two unknown quantities 
in the antigen titer, the syphilitic serum and the antigen to be titered). 
The dose, 0.5 cc. of a 1 : 10 dilution, which was selected because it was 
less than one-third of the anticomplementary unit, contains more than 
two antigenic units (the antigenic unit being 0.2 cc). Part IV shows 
that the antigen is specific, since 0.5 cc. of a 1 : 10 dilution (the amount 
decided upon for use in the reactions), does not l^ind complement in 
the presence of 0.1 cc. of normal serum (tube No. 16). Of course the 
antigen should give negative results with a large number of normal 
specimens before it is actually employed in doing diagnostic reactions. 
The serum controls (Part V) are set up with a standard antigen to make 
certain that the normal serum employed is negative, (tube No. 19), 
and that the diluted syphilitic serum (tube No. 17) actually completely 
fixes the complement in the dilution employed. The latter is a cross 
check on the accuracy of the unit titer of the syphilitic serum. Tubes 
Nos. 18 and 20 are set up to correspond with the rear-tube control of 
the Wassermann reaction to show that neither positive nor negative 
serum has anticomplementary properties sufficient to interfere with the 
reaction. The result should be as indicated in the table. 

To summarize, the amount of antigen employed in the test should 
be one-third of the amount which is anticomplementary or less; it 
should contain at least two antigenic units; and it should be less than 
one-half of the amount which hemolyzes sheep cells. 



THE WASSERMANN REACTION 147 

Dilution of Antigen.— When the antigen has been titrated and its 
properties ascertained, the worker is ready to determine the dilution. 
This to be made so that the required amount is 0.5 cc. of the emulsion. 
It is desirable to make the emulsion as strong as possible keeping it 
within the limits imposed by its anticomplementary titer (see above). 
In making the dilution, the required amount of physiological saline 
solution is measured out in a cylindrical graduate and the alcoholic 
antigen is added to it slowly, a drop or two at a time, mixing well after 
each addition of antigen. Other workers, Ruediger for example, advo- 
cate adding the salt solution slowly in small quantities to the antigen. 
The essential point is to follow the same method in diluting antigen 
for titration and for the reactions proper. 

General Cautions.— In doing the reactions the worker must guard 
against non-specific complement-fixation. The hemolytic system 
should be carefully balanced. The human serum must be carefully 
inactivated, and a reading of the reaction should be not made unless 
the rear control tube for the reaction in question be clear. Positive 
controls should be positive and negative controls should be negative. 
An antigen may act queerly after a period of some months, either losing 
in strength or giving partial "false positives" on account of increased 
anticomplementary qualities. Mistakes on account of this may be 
avoided if all reactions are set up with three or four antigens, made at 
different times. The various properties of a constantly used antigen 
should be tested out by occasional titers. 

It is not necessary to titer the antigens each time reactions are done, 
although the prescribed anticomplementary controls must be set up. 

Controls and Preliminary Set of Reactions.— The directions given 
contemplate the use of an "indicator" (hemolytic) system made up of 
1.5 units of complement and 2 units of amboceptor. Some workers 
recommend using 2 units of complement and 2 units of amboceptor 
when the cold fixation is used. This is probably the conservative 
Course especially with cholesterolized antigens. The use of one unit 
of each reagent is unwise, since an excess of hemolytic substance is 
needed to overcome the anticomplementary action of human serum 
and antigen. 

In performing Wassermann reactions, the use of reliable controls 
is absolutely essential. When work is being done regularly, this is 
not particularly difficult, since specimens may be kept from one 
reaction day to another. It is advisable to be certain that the negative 
control is really from a normal person. Specimens carried over from 
the preceding week should be again subjected to 56° C. for fifteen 
minutes to destroy any anticomplementary substances which may have 
developed on standing. An obviously infected serum should not be 
employed, since it may give a falsely positive result. Cloudiness of 
serum is sometimes due to high lipoid content. When the tube is 
held for a few moments in warm water, the opacity due to lipoids 
clears up, that due to bacteria remains. An especially desirable control 



148 EXAMINATION OF THE BLOOD 

is a positive spinal fluid. Spinal fluid keeps indefinitely if sterile, its 
antibody content does not change, and anticomplementary sub- 
stances are not developed. A syphilitic unit titer should be made in 
the manner to be described later under examination of the spinal fluid 
(page 151) and this should be repeated as a control before setting up 
the day's reactions. With a properly balanced hemolytic system, the 
unit should be the same. When the preliminary reactions are set up, 
they should then include at least one positive and one negative blood 
serum from the preceding day's work, a positive spinal fluid of known 
titer, the antigen controls, and if possible a serum which gave a faintly 
positive result. 

The antigen controls consist of two tubes for each antigen, one with 
double the amount and one with three times the amount of antigen 
used in the reactions. To each is added 2 units of complement, and 
after incubation, 2 units of hemolytic amboceptor and the regular 
amount of sheep corpuscles. The tubes are set up as directed below 
for the reactions proper. If the proper balance has been secured 
between the members of the hemolytic "indicator" system, the 
results should be the same as on the preceding day's work, i. e., the 
negative should be negative, the strongly positive positive, the faintly 
positive as before, the spinal fluid giving a positive reaction with the 
same quantity as previously, and all the antigens controls showing 
complete hemolysis. If the formerly strongly positive reaction is not 
so strong, if the spinal fluid shows a negative reaction with the amount 
which should be positive, and a positive only with a high amount, and 
the blood (formerly faintly positive) is negative, then it is apparent 
that the complement is too strong, and its strength must be reduced. 
If, on the other hand, the negative blood gives a ± or a one plus result 
in any or all tubes, if the blood formerly faintly positive is strongly 
positive, if the spinal fluid shows complement-fixation with smaller 
quantity of fluid than usual, and if the antigens controls showed some 
impairment in hemolysis, the complement has been made too weak. 
The spinal fluid is the best index. Blood serum is more complex and 
occasionally the development of anticomplementary substances will 
render negatives falsely positive or, on the other hand, the deteriora- 
tion of antibody due to preservation or re-inactivation allows positives 
to react negatively. It will be seen that the worker must interpret 
all his evidence judiciously and act accordingly. It is only by carrying 
out this preliminary work that the worker can do work, the results of 
which check from week to week. 

Performance of the Reactions Proper.— A series of racks with double 
row of holes are prepared. If three antigens are to be employed, three 
tubes will be needed in the first row, and one control in the back row 
for each reaction. In each of the tubes in the front row is placed 
0.5 cc. (containing 0.1 cc. undiluted serum) of the diluted inactivated 
serum to be tested, and 1.0 cc. (containing 0.2 cc. of undiluted serum) 
in the rear control tube (Table F) . This is repeated for each serum to 



THE WASSERMANN REACTION 



149 



be examined. Positive and negative control sera are set up in the same 
way, and the antigen controls are-prepared at the same time (page 146) . 
Then to the tubes in front and back rows alike, is added 0.5 cc. (1.5 
units) of complement dilution. In the front row of the reaction tubes 
is placed 0.5 cc. of antigen dilution, the first antigen being placed in the 
first tube of each reaction, the second in the second tube, etc. If 
warm fixation be employed, the racks are now placed in the water-bath 
at 37° C. for one hour, shaking occasionally (Fig. 36). If cold 
fixation be employed, they are kept at a temperature of 8 to 12° C. for 
four to twelve hours. When the required time has elapsed, into each 
tube, front and back alike, is added 0.5 cc. of hemolytic amboceptor 
dilution and 0.5 cc. of sheep corpuscle suspension and the racks are 
placed in the water-bath at 37° C. for one hour or until the antigen 
controls, the rear tube controls, and the negative serum controls have 
hemolyzed completely. It is important that during this time they be 
shaken frequently to prevent settling of the corpuscles to the bottom 
of the tubes. 



TABLE F.— WASSERMANN REACTION ON BLOOD SERA. 









A mount 
of 

serum 
dilution 

(1:5), 




Amount 




Amount 


Amount 












Amount 


of com- 




of ambo- 


of sheep 






Tube 

No. 


Serum 
used. 


Purpose 
of tube. 


of 
antigen 
dilution 


plement 
dilution 
(0.5 cc. = 




ceptor 
dilution 
(0.5 cc. = 


corpus- 
cle emul- 
sion (5 




Result 
should be. 








cc. 


1.5 un.), 




2 units) , 


per ct.), 
















cc. 




cc. 


cc. 






1 . . 


A 


For test 


0.5 


0.5 


0.5 




0.5 


0.5 




? 


2 




A 


Control on 
A test 


1.0 





0.5 


3 


0.5 


0.5 


3 


C.H. 1 


3 




B 


For test 


0.5 


0.5 


0.5 


A 


0.5 


0.5 


A 


? 


4 




B 


Control on 
B test 


1.0 





0.5 


O 


0.5 


0.5 



O 


C.H. 


5 




C 


Positive 
control 


0.5 


0.5 


0.5 


6 


0.5 


0.5 


Q 


+ + + + 


6 




C 


Control on 
C serum 


1.0 





0.5 


CO 


0.5 


0.5 


CO 


C.H. 


7 




D 


Negative 
control 


0.5 


0.5 


0.5 


1 


0.5 


0.5 


A 


C.H. 


8 




D 


Control on 
D serum 


1.0 


.0 


0.5 


,0 

CD 


0.5 


0.5 


A 


C.H. 


9 




None 


Control on 
antigen 





1.0 


0.5 


a 


0.5 


0.5 


1 


C.H. 


10 




None 


Control on 





1.5 


0.5 




0.5 


0.5 




C.H. 






antigen 



















This table provides for the use of only one antigen. When more are employed, addi- 
tional front tubes (corresponding to 1, 3, 5 and 7) should be set up for each serum. 
Only one control tube (corresponding to 2, 4, 6 and 8) will be required for each serum. 
Two antigen control tubes (corresponding to 9 and 1 0) should be set up for each antigen 
employed in the test. 

Detection of Native Antisheep Amboceptor. — If desired, each specimen 
of serum may be tested to determine the presence of native antisheep 

1 C.H. means complete hemolysis; + + + +, no hemolysis. 



150 EXAMINATION OF THE BLOOD 

amboceptor during the first hour that the reactions are in the incu- 
bator or refrigerator. 

In each of a series of tubes is placed 0.25 cc. of a 1 : 5 dilution of human 
blood sera upon which Wassermann reactions are to be done and 0.25 
cc. of complement dilution (one-half amount used for reactions), 0.5 cc. 
of cell emulsion, and 1.5 cc. normal saline. Place tubes in the incu- 
bator or water-bath at 37° C. for thirty minutes, shaking two or three 
times. When complete hemolysis results, it is evident that the serum 
contains enough native antisheep hemolytic amboceptor to complete 
the reaction so no further hemolytic amboceptor is added to the tubes 
in the reaction proper. By taking this simple precaution, one avoids 
the possibility of adding a great excess of unnecessary amboceptor. 
The native antisheep amboceptor may be removed from the serum if 
desired. 

Removal of Native Antisheep Amboceptor. — To each 1 cc. of inacti- 
vated human serum is added 0.1 cc. of undiluted washed sheep cells. 
The mixture is shaken well, placed in the water-bath at 37° C. for 
thirty minutes, and then centrifugalized and the serum removed from 
the sedimented cells. The serum may be regarded as undiluted. 

Reading the Reactions.— A preliminary reading may be taken as soon 
as the reactions are completed, at the end of the second incubation 
period. A final report, however, on these readings would be unsafe 
unless one has had much experience in such work. 

A frank negative may be read at once. If an immediate report on 
any specimen is imperative, a doubtful reaction should be centrifugal- 
ized in order that the cells may be brought to the bottom of the tubes 
and the supernatant fluid be left cell-free for the detection of shades of 
hemolysis. The final readings are made usually after the reactions 
have stood two or three hours to permit partial settling of the cells. 
It is often convenient to allow them to stand in the refrigerator over 
night. Several criteria must be fulfilled before the reactions can be 
regarded as satisfactory. The positive control should give a positive 
result and the negative should be negative. All of the antigen controls 
must show complete hemolysis, and there should be almost universal 
complete hemolysis in the rear-tube controls. An occasional rear tube 
may fail to show hemolysis. This would be due probably to a poor 
specimen of serum, but if any marked number showing this condition, 
it is an indictment against the technic. When hemolysis is not com- 
plete in a rear-row control tube, that particular reaction must be dis- 
regarded, and another specimen of blood should be obtained from the 
patient. 

In reading the results, the clarity or tinging of the supernatant fluid 
should be observed and the amount of residual sedimented cells noted. 

The scale of readings is purely arbitrary. The writer employs one 
based on + + + representing a strongly positive result. The scheme 
proposed by Citron, however, is widely employed and will be given here. 
+ + + + represents complete fixation of complement. There is no 
hemolysis. The result is strongly positive. 



THE WASSERMANN REACTION 151 

+ + + represents 75 per cent, or more of complement-fixation, or 25 

per cent, or less of hemolysis. The result is said to be 

positive or moderately positive. 
+ + represents 50 to 75 per cent, of complement-fixation, or 25 to 

50 per cent, of hemolysis. The result is said to be faintly 

positive. 
+ represents 25 per cent, or less of complement-fixation or more 

than 75 per cent, of hemolysis. The result is said to be 

very faintly positive. 
=±= represents a faint rim of red cells only at the bottom of the 

tube. This is a doubtful. (It is usually negative.) 
— represents complete hemolysis. The result is negative. 
Wassermann Reaction on Spinal Fluid.— Since spinal fluid is free 
from complement and anticomplementary substances, inactivation is 
unnecessary. If free from blood and taken under sterile conditions in 
a sterile tube, it may be preserved in a refrigerator for a considerable 
period of time with no alteration in its antibody content and without 
the development of anticomplementary substances. Bacterial con- 
tamination, of course, will interfere with proper results. 

In early work with the Wassermann reaction, the mistake was made 
of employing small quantities of spinal fluid, that is, it was used in the 
same amount as was blood serum. Larger quantities may be employed 
safely; and with them a much greater number of positive results is 
obtained in cases with definite syphilitic involvement of the central 
nervous system. 

Measurement of the syphilitic antibody content of spinal fluid is 
extremely easy and should be done at least in all cases where treatment 
for cerebrospinal syphilis is contemplated, because observation of the 
change in the titer under treatment affords an index to progress. A 
series of tubes is set up, six in the front row of the test-tube rack and one 
in the rear. Into the front row tubes are placed respectively, 0.1 cc, 
0.2 cc, 0.4 cc, 0.6 cc, 0.8 cc, and 1.0 cc, of spinal fluid (Table G). 
In the rear control tube is placed 2.0 cc. If there is insufficient fluid 
for all these tubes, the higher doses should be employed, always 
reserving enough for the rear control tube which should double the 
amount of the largest dose employed in the front row. To each front 
tube is added 0.5 cc. of antigen dilution and then to all tubes is added 
0.5 cc. of complement dilution (containing 1.5 units). Physiological 
saline solution is added when necessary to bring the final volume to 
2.5 cc Fixation is carried out as with blood serum, either in water- 
bath or incubator at 37° C, for one hour, or at 8 to 12° C. from four 
to ten hours. Then 0.5 cc. of amboceptor dilution (containing 2 units) 
and also 0.5 cc. of 5 per cent, sheep corpuscle emulsion are added to 
each tube, front and back. The tubes are kept in the water-bath at 
37° C. for one hour with frequent shaking, or until the negative 
controls, the antigen controls, and the rear-tube controls have entirely 
hemolyzed. In reading and reporting, the tube should be noted in 



152 



EXAMINATION OF THE BLOOD 



which a positive result first appears. In the illustrative table giving 
hypothetical results, the report would be ++ with 0.4 cc, and 
+ + + + with 0.6 cc. The rear control must show complete hemolysis 
or the result will have to be considered unsatisfactory. 

TABLE G.— WASSERMANN REACTION WITH SPINAL FLUID. 









Amount 


Amount 




Amount 


Amount 










Amount 


Amount 


of com- 


of 0.9 




of ambo- 


of sheep 








Tube 
No. 


of 


of 


plement 


per cent. 




ceptor 


corpus- 




Hypo- 




spinal 
fluid, 


antigen 


dilution 


NaCl 




dilution 


cle emul- 




thetical 


Readings. 


dilution, 


(0.5 cc. = 


solution, 




(0.5 cc. = 


sion (5 




results. 






cc. 


cc. 


1.5un.), 
cc. 


cc. 




2 units), 
cc. 


per cent, 
cc. 








1 . . 


0.1 


0.5 


0.5 


0.4 


*■ 3 


0.5 


0.5 


*s| 


C.H.i 


_ 


2 . . 


0.2 


0.5 


0.5 


0.3 


rf)^ 


0.5 


0.5 


•5*° 


C.H. 


— 


3 . . 


0.4 


0.5 


0.5 


0.1 


|§ 


0.5 


0.5 


"° o 


S.H. . 


+ + 


4 . . 


0.6 


0.5 


0.5 







0.5 


0.5 




N.H. 


+ + + + 


5 . . 


0.8 


0.5 


0.5 





"S 1 " 


0.5 


0.5 


"£"" 


N.H. 


+ + + + 


6 . . 


1.0 


0.5 


0.5 







0.5 


0.5 


£6 

CO 


N.H. 


+ + + + 


Control 


2.0 





0.5 





0.5 


0.5 


C.H. 





Interpretation of Results.— It is highly unfortunate that all workers 
do not employ a technic which has been standardized in its funda- 
mental requirements. At present there are almost as many variations 
of technic as there are workers. It is incumbent upon the clinician, 
therefore to know something about the technic which is being employed 
in work done for him before he attempts to interpret the results, and it 
behooves the laboratory worker to criticize his technic impartially, 
to study the possible sources of error, to eliminate them whenever 
possible, and above all, to so standardize his method so that it will 
give constant and comparable results. In case of doubtful reaction, 
it is much fairer to clinician, laboratory worker, and especially to the 
patient to secure a second or even a third specimen, than to give an 
erroneous report. 

Interpretation of the results should not be attempted by the labora- 
tory worker unless he is able and willing to go into the clinical evidence. 
It is even more important that the clinician should be well informed as 
to the significance of the laboratory results and that he should be able 
to interpret the variations from the expected. 

A negative result absolutely does not mean that syphilis can be 
excluded, as will be seen by a study of the table (page 153). In 
primary syphilis the reaction does not appear until the human organism 
is able to develop antibodies. This time varies with different patients, 
occurring occasionally as early as the third or fourth day after the 
appearance of the chancre and sometimes not until secondary signs 
are in evidence. In a lesion which arouses the suspicion of being 
a chancre, the diagnostic method of choice is the detection of the 

1 C.H. means complete hemolysis; S.H., slight hemolysis; N.H., no hemolysis. 



THE WASSERMANN REACTION 



153 



Treponema pallidum, for treponemata may be found in about 80 per 
cent, of cases of primary syphilis, while the Wassermann reaction gives 
positive results in only about 53 per cent, of cases. Even in the 
secondary stage, when the highest percentage of positive results is 
obtained, occasional negative results are seen, occurring when the 
patient is in a debilitated condition, or uses alcohol to excess. In 
definite tertiary syphilis, a considerable percentage of negative results 
is reported. This may be accounted for by the diminished resistance 
on the part of the patient, alcoholism or recent active treatment. 
Another factor which explains many negative reactions in late cases 
is the localization of the lesions in portions of the body not especially 
accessible to the general circulation or the partial walling-off of gum- 
matous deposits. 

TABLE SHOWING THE PERCENTAGE OF POSITIVE WASSERMANN 

REACTIONS OBTAINED BY DIFFERENT WORKERS IN 

DIFFERENT STAGES OF SYPHILIS. 



Primary. 



Secondary 



Tertiary. 



Tabes 
dorsalis. 



Congenital. 



Boas's collected 
cases 1 

Fildes and Mc- 
intosh 2 . 

Craig 3 . 

Swift* . . . 

Noguchi 5 . 



loco 
33 



1173 

85 



100 

100 
80.0 



The fallacy of placing sole dependence upon a blood serum Wasser- 
mann reaction is most apparent in syphilis of the central nervous 
system, where blood-serum reactions are often negative while unmis- 
takable evidence of syphilis is found when the spinal fluid is examined. 

Not infrequently patients who are under treatment for syphilis have 
the impression that a single negative Wassermann reaction means that 
they are cured. This is unfortunate, since a single negative under such 
circumstances means absolutely nothing. 

The possibility of error in interpreting a strongly positive report is 
not so great. In the temperate zone a strongly positive reaction ordi- 
narily means syphilis. While there are occasional exceptions, they 
usually occur in cases where there would be small possibility of clinical 
confusion, such as leprosy, and in the tropics, frambesia. 

1 Die Wassermannsche Reaktion mit bes. Beriicksichtigung ihrer Klinischen Verwertbarkeit, 
2d edition, Berlin, 1914. (Quoted in Special Report Series, No. 21, Medical Research Com- 
mittee, London, 1918, p. 6.) 

2 Brain, London, 1913, xxxvi, 193. 

3 Am. Jour. Med. Sc, 1915, cxlix, 41. 
* Arch. Int. Med., 1909, iv, 374. 

6 Serum Diagnosis of Syphilis, J. B. Lippincott Co., Philadelphia, 1911. 



154 EXAMINATION OF THE BLOOD 

Faintly or partially positive results, however, should be interpreted 
with even greater caution, since either may be seen occasionally in 
tuberculosis and cancer. With indefinite results, repetition of the test 
is highly advisable. A plus-minus result should never be interpreted 
as a positive, especially when obtained with cholesterolized antigens. 

The Reaction in Treated Cases.— When treatment is started early in 
the course of spirochetal invasion and pushed intensively, it often 
produces the prompt reversal of a positive to a negative but with 
cessation of treatment the test again becomes positive. In cases of 
longer standing where time has permitted a more generalized invasion 
of the body-, and where the infection is well seated, a positive reaction 
is often rendered negative with the utmost difficulty, even with the 
most intensive treatment by arsphenamine and mercury. Indeed in 
some cases, referred to as "Wassermann fast," a negative seems 
unobtainable. 

The conversion of a positive result to negative under treatment may 
be regarded as a favorable sign but treatment should not be stopped 
because of a random negative Wassermann reaction until the history 
of the case and accumulated clinical and serological experience shows 
that a prolonged intermission for rest from treatment and for observa- 
tion is warranted. 

Serological Criteria cf a Cure in Syphilis.— To pronounce a patient 
as cured upon serological grounds alone is not good clinical practice. 
The entire history must be considered, special consideration should be 
paid to the recency or remoteness of the infection, the promptness with 
which treatment was started with relation to the primary infection, 
the amount, duration, and intensity of treatment, the occurrence of 
so-called "tertiary" lesions and of signs and symptoms pointing to 
involvement of the central nervous system or of the aorta. Of equal 
importance is the searching physical examination of the patient with 
particular reference to mouth and throat, glands, aorta and central 
nervous system. As a third method of examination, the laboratory 
offers exceptional resources, yielding valuable evidence, possibly unob- 
tainable in any other way. The laboratory side should include three 
types of examination: 

1. Repeated Wassermann reactions upon the blood serum. 

2. Provocative Wassermann reactions. 

3. Examination of the spinal fluid. 

1. Series of Wassermann Reactions on the Blood.— When clinical 
experience shows that it will be safe to permit a prolonged intermission 
in treatment, this may be stopped and the Wassermann reaction done 
with the blood serum at intervals of three months. If any test be 
positive, treatment should be resumed. If all are negative, the pro- 
vocative test should be made. 

2. Provocative Test— It has been shown that in some cases of 
latent syphilis showing negative results repeatedly, a positive is 
obtained after the intravenous injection of small doses of arsphenamine. 



THE WASSERMANN REACTION 



155 



This may be explained by assuming that the arsenical preparation 
destroys spirochetes on account of its power of permeation and its 
consequent ability to be carried to out-of-the-way foci, and that the 
liberation of toxins leads to the elaboration of syphilitic antibodies, 
which give a positive result. Whatever the explanation may be, the 
fact remains that a number of cases which have been treated to the 
point where repeated negatives have been secured, respond positively 
after a provocative dose of arsphenamine. 

The technic is simple, involving only the intravenous injections of 
arsphenamine (0.2 to 0.3 gm.) or neo-arsphenamine (0.4 to 0.5 gm.), 
and securing specimens of blood at two-day intervals for ten days. 

3. Examination of Spinal Fluid.— It is the general feeling among 
modern syphilologists that the spinal fluid should be examined, cer- 
tainly before the patient is regarded as cured, and at other times in the 
course of infection if indications arise. The technic for obtaining 
spinal fluid will be given in a later section (page 371). The examina- 
tion should include cell- count, globulin determination, Lange colloidal 
gold curve, as well as Wassermann reaction. 

Laboratory Diagnosis of Syphilis of the Central Nervous System.— What- 
ever has been said regarding the folly of excluding syphilis on the 
strength of a negative Wassermann reaction obtained with blood 
serum applies with added force to the diagnosis of any form of syphilis 
of the central nervous system. This is evident from a cursory examina- 
tion of the following table. The large majority of cases of tabes 
dorsalis, paresis, and syphilitic meningo-endarteritis give some indica- 
tion in the spinal fluid which will assist in making a diagnosis, if not 
a positive Wassermann reaction, an increased cell-count, increased 
globulin, or specific curve with the Lange colloidal gold test. 

TABLE SHOWING RESULTS OF LABORATORY TESTS IN DIFFERENT FORMS 
OF CEREBROSPINAL SYPHILIS. 1 PERCENTAGE OF POSITIVE RESULTS. 











Blood, cerebrospinal fluid 














£ 


Wassermann reaction. 






S 






izU 












a 


a 


M 




































§3 

1 


S.S 
P 


ga 


a .2 


do 


doq 


o 


§2 


°d 


§Z 




d t. 
















o~-^ 




£ 


F 


o 


p 


o 


o 


o 


O 


o 


o 


Cerebrospinal 






















syphilis . 


21 


62 


86 


100 


94 


88 


88 


81 


33 


25 


Tabes dorsalis . 


30 


66 


97 


87 


86 


83 


69 


55 


23 


4 



Reactions done with half quantities of all reagents. Amounts of spinal fluid used 
by authors shown above, and below, in parenthesis, the amount of which this corresponds 
in system employed by Hauptmann, Nonne and others. 



From Ellis and Swift, Jour. Exper. Med., 1913, xviii, 162. 



156 EXAMINATION OF THE BLOOD 

Summary.— In the primary stage the diagnostic value of the Wasser- 
niann reaction is limited, increasing in value as the infection progresses, 
but the diagnostic method of choice is the search for the Spirocheta 
pallida, which should be performed routinely in all suspicious sores. 
In the secondary stage, the Wassermann reaction is most constant, 
since the positive results approach 100 per cent. In the so-called 
tertiary stage, with obscure manifestations, visceral or bony-system, 
the reaction is valuable, especially in differential diagnosis, but only 
about 80 to 90 per cent, of positive results are obtained. In central 
nervous system syphilis, the blood-serum reaction may be misleading 
unless judiciously interpreted, since only about 66 per cent, of cases of 
tabes dorsalis give a positive reaction. In such cases the spinal fluid 
examination is very much more dependable. In congenital syphilis 
the percentage of positive is in the neighborhood of 90 to 95 per cent. 
The reaction is of much importance in children with syphilitic parents, 
because of the long period of latency seen in many cases. It is par- 
ticularly useful in cases where adoption is contemplated and no history 
of the parents obtainable. Treated cases must be carefully considered 
on their own merits after complete survey of history, physical findings, 
and laboratory examinations. 

The Gonococcus Complement-fixation Test.— The general theoretical 
principles underlying the complement-fixation for gonorrhea are the 
same as those underlying the Wassermann reaction. In practice the 
antigen is truly specific. The directions which have been given in 
the description of the Wassermann reaction for preparing and titra- 
ting the complement and hemolytic amboceptor and for making 
the emulsion of sheep cells, together with those regarding preliminary 
reactions and controls, apply to the gonococcus-fixation tests. In 
most laboratories where the latter tests are carried out, Wassermann 
reactions are done also and it is a simple matter to use the same set of 
reagents. 

The Antigen.— A polyvalent antigen should be used, preferably one 
containing ten of the strains shown by Torrey to be serologically 
distinct. The gonococcus may be grown upon the medium described 
bj- Thomson or upon ascitic or hydrocele-fluid agar. Stock cultures 
must be transplanted every forty-eight hours, and the cultures must 
be kept constantly at 37.5° C, since variations in temperature will 
result in poor growth. To prepare the antigen take stock cultures 
twenty-four hours old and make cultures on tubes 1 by 6 inches 
containing Thomson's medium. These tubes should be incubated 
twenty-four hours and the growth then transferred to wide-mouthed 
Blake bottles containing the same medium, planting into each Blake 
bottle the bacteria from one tube, using a sterile cotton swab for 
inoculating. The Blake bottles are incubated twenty-four hours and 
the growth is washed off with sterile, neutral distilled water, using from 
5 to 10 cc. of water to each bottle, the amount depending upon the 
growth. The water is not allowed to remain on the agar for more than 



OTHER SERUM REACTIONS 157 

a few seconds. The suspension is then autolyzed by placing the 
container in a water-bath, first at 56° C. for one hour and then at 80° C. 
for one hour. The autolyzed emulsion is filtered through a Buchner 
filter well packed with paper pulp and then through a neutral, sterile 
Berkefeld filter of N or V porosity. 1 

After filtration the antigen should be bottled under aseptic pre- 
cautions and sterilized on three successive days at 56° C. for thirty 
minutes on each day. It should be kept in the refrigerator. A 
preservative may be added, 0.1 cc. of a 1:100 dilution of phenol to 
each cc. of antigen. 

Of late, the writer has secured excellent results with the antigen 
prepared by the Parke-Davis Company, especially with the cold 
fixation method. 

Titration of the Antigen. —The antigen made according to the fore- 
going directions should be kept in the refrigerator. It is a distilled 
water extract of gonococci. Just before use, the desired quantity is 
placed in a test-tube and made isotonic by adding to nine parts of the 
watery extract one part of sterile 9 per cent, salt solution. The salt 
solution should not be added until the day when the antigen is to be 
used, since it interferes with its stability. The extract which has been 
made isotonic is now diluted by adding to one part of the extract nine 
parts of sterile normal salt solution. 

The commercial antigen referred to is diluted with nine parts of 
normal salt solution when employed. 

A series of test-tubes is set up. Into these are placed respectively 
2.0 cc, 1.8 cc, 1.5 cc, 1.2 cc, 1.0 cc, 0.8 cc, 0.6 cc, 0.4 cc, 0.2 cc, and 
0.1 cc, of the 1 : 10 dilution of antigen. . With a new and untried anti- 
gen, the quantities may have to be increased or diminished and several 
trials may be necessary to determine the limits of anticomplementary 
power. 

The hemolytic system is adjusted as described in the section on the 
Wassermann reaction. Complement dilution is added to each tube, 
the dilution having been made so that 0.5 cc. of the dilution contains 
1 .5 units of complement, as shown by previous titration of the comple- 
ment. Normal salt solution is added to each tube when needed to 
bring the total volume to 1.5 cc. The tubes are subjected to a tempera- 
ture of 37° 0. for one hour or to a temperature of 8 to 12° C. for four to 
twelve hours, at the end of which time there are added to each tube 0.5 
cc of 5 per cent, suspension of sheep corpuscles and 0.5 cc. of ambocep- 
tor dilution, the latter so made up that this quantity contains 2 units. 

1 Since new Berkefeld filters are often very alkaline, they should be taken apart and 
boiled in distilled water for five minutes, scrubbed with a small brush in fresh water, 
and again boiled in distilled water, repeating the process three times. Then the filter 
may be set up and hot water allowed to stand in it for five minutes, after which neutral 
distilled water is allowed to run through until the fluid tests neutral with phenolphtha- 
lene. All parts may be sterilized in distilled water in the Arnold sterilizer before use. 
After use the filter should be boiled in distilled water and scrubbed. It may be attached 
to the filtering flask and sterilized ready for use. 



158 



EXAMINATION OF THE BLOOD 



The tubes are again incubated for thirty minutes at 37° C. The tube 
which shows beginning inhibition of hemolysis is said to contain the 
anticomplementary unit, and less than one-half of this amount should 
be used in the test. The antigen should be added to the tubes in this 
quantity for the reaction. If, for example, the tube in the titer which 
contained 1.8 cc. was entirely clear while the tube containing 2.0 cc. 
of the antigen showed some inhibition of hemolysis, we might employ 
0.9 cc. of the diluted antigen in the reaction. 

Performance of the Reaction.— As a control, there should be at hand 
either the serum of a rabbit who has been immunized with gonococci 
or the serum of a patient which has given a gonococcus-fixation test 
previously. If rabbit serum is used as a control, it should be deter- 
mined before immunization that the rabbit is not one which gives 
non-specific complement-fixation in the presence of bacterial extracts. 
Known normal serum must be used as a control. The reaction may be 
set up according to the table. 

GONOCOCCUS COMPLEMENT-FIXATION TEST. 



Tube 


Patient's 


Amount 
of com- 
plement 


Amount 
of anti- 
gen di- 
lulion 


Amount 
of 0.9 

per cent. 
NaCl 

solution, 
cc. 




Amount 
of ambo- 
ceptor 


Amount 

of 5 
per cent. 

sheep 
corpuscle 
emul- 
sion, 
cc. 




Hypo- 




No. 


serum, 
cc. 


dilution (0.5 cc. = 
(0.5 cc. = j anti- 
1.5 un.), j comp. 
cc. i unit), 
cc. 




dilution 

(0.5 cc. = 

2 units), 

cc. 




thetical 
results. 


Readings. 




Unknown 








o . 












1 


0.1 


0.5 


0.5 


0.4 


|3 


0.5 


0.5 


i 

o 


? 


? 


2 


0.2 
Normal 


0.5 





0.8 


■«J§ 


0.5 


0.5 


A 

c 
o 


C.H.i 




3 


0.1 


0.5 


0.5 


0.4 


°i 


0.5 


0.5 


£. 


C.H. 


Negative. 


4 


0.2 
Positive 


0.5 





0.8 


d-2 


0.5 


0.5 


6 


C.H. 


Strongly 


5 


0.1 


0.5 


0.5 


0.4 


""*" 


0.5 


0.5 


a 


N.H. 


positive. 


6 


0.2 


0.5 





0.8 


• a « 


0.5 


0.5 


■5 

03 


C.H. 




7 




0.5 


0.5 


0.5 


2J 


0.5 


0.5 




C.H. 


Antigen 
control. 


8 




0.5 


1.0 





0.5 


0.5 


£ 
5 


C.H. 


Antigen 












a — 








control. 



Clinical Value of the Gonococcus Complement-fixation Test— The 
principal objection to the complement-fixation test for gonorrhea is 
the difficulty which is encountered in preparing the antigen. This 
arises from the fact that a number of strains of the gonococcus must 
be kept alive by frequent transplantation and that the media which are 
used for this organism are troublesome to prepare. 

It is rather generally agreed that the reaction is specific in that 
positives are not given in any condition except gonococcus infections. 
The antibody, however, is often not present in large quantities even in 

1 C.H. means complete hemolysis; N. H., no hemolysis. 



OTHER SERUM TESTS 159 

definitely gonorrheal infections, because the process may be a limited 
one. This reduces the possibility of securing positive reactions when 
infection is present. The reaction is rarely present in the first four to 
six weeks of a urethritis. Kolmer states that in exacerbations of a 
chronic urethritis, a positive reaction is secured in about 80 per cent, 
of the cases, that the reaction is positive in from 30 to 40 per cent, of 
the cases with mild involvement of the prostate gland complicating 
chronic urethritis, and that from 50 to 80 per cent, of positive results 
are secured in cases of chronic urethritis with marked involvement of 
the prostate gland and epididymitis. A positive reaction is secured 
in about 80 per cent, of the cases of gonorrheal iritis. 

A negative result cannot be interpreted as excluding gonorrheal 
infection, while a positive result is usually taken as pointing to the 
presence of an active focus. In women the reaction is usually negative 
until the infection has reached the cervical canal, or Bartholin glands, 
while with children it is often positive with a severe vulvovaginitis. 
Nearly 100 per cent, of cases with gonorrheal arthritis show positive 
complement-fixation tests. It must be remembered that the adminis- 
tration of antigonococcus serum or gonococcus vaccine will always 
be followed by a positive reaction in a patient who has recently had 
gonorrhea. Schwartz states, however, that the administration of 
gonococcal vaccines or sera to a person who has never had gonorrhea 
will not produce a positive reaction in his blood. 

Weakly positive reactions are usually interpreted as pointing to an 
approaching cure, and if the test is repeated after some weeks, it nearly 
always becomes negative unless meanwhile vaccines have been given 
or a new infection acquired. 

Clinically, one of the chief uses of the complement-fixation test is in 
the investigation of completeness of cure in candidates for marriage. 

The value of the test is brought out by the fact that in 30 per cent, 
of cases in which no clinical evidence of disease persists, a positive 
reaction points to the persistence of an unsuspected focus. 

Complement-fixation Test in Tuberculosis.— The complement-fixation 
test in tuberculosis cannot be said to have an established clinical value. 
A number of methods have been proposed, the essential differences 
being in the manner of preparing the antigen, and encouraging reports 
have been made by the proponents, but unfortunately others have been 
unable to secure equally good results in employing the same methods. 
While the test is of distinct interest from a scientific standpoint, the 
technic will have to be perfected before it can become a generally 
useful diagnostic measure. The antihuman or antisheep hemolytic 
system may be used and the strength of the reagents should be balanced 
by careful titration and by use of preliminary sets of reactions as has 
been described (see page 147). The following methods have been 
proposed : 

Craig's Method.— The antigen is a modification of Besredka's and is 
an alcoholic extract of several strains of the tubercle bacillus, grown 



160 EXAMINATION OF THE BLOOD 

on a liquid medium composed of alkaline bouillon to which has been 
added a teaspoonful of egg white and egg yolk for each 250 cc. of 
bouillon. After growth has proceeded for some time, the bouillon is 
mixed with an equal quantity of 95 per cent, alcohol and the mixture 
is shaken in a shaking machine for twelve hours, after which it is 
allowed to stand in the incubator at 37° C. for twenty-four hours. 
It is then shaken for six hours, again incubated at 37° C, and filtered 
through very fine filter paper or a Berkefeld filter. The filtrates of 
various strains should be mixed together and should be tested for 
anticomplementary power and hemolytic properties. The antigen must 
be stored in the ice-box. 

Miller and Zinsser's Method.— Tubercle bacilli are grown upon 
suitable solid media. The growth is scraped off. The bacilli may be 
killed with heat or not as desired. Twenty milligrams of the moist 
tubercle bacillus mass are weighed out, placed in a conical 15 cc. 
centrifuge tube with 90 mg. sodium chloride. The paste is ground 
by hand for one hour with a glass rod. Distilled water is added to the 
point of isotonicity (i. e., 10 cc. should be added to the quantities given 
here). The antigen is shaken just before using and the heavier 
particles are allowed to settle out for a few moments. The resulting 
emulsion should be titrated for anticomplementary and antigenic 
properties. This antigen utilizes the endotoxins extracted from the 
bacilli. The writer has failed to obtain with this antigen the high 
percentage of positive results reported by Miller and Zinsser. 

Petroff' s Method.— Petroff advocates the use of more than one 
antigen, and suggests three different methods of preparation: 

Antigen I is polyvalent, and is made from six strains of human 
bacilli and from one of bovine tubercle bacilli. They are grown on 
potato broth. 

The potato broth is prepared by infusing 500 grains of grated potato 
in 100 cc. of distilled water over night in the refrigerator. The infusion 
is then filtered from the pulp through gauze. It is titrated and the 
reaction is adjusted to 1 per cent, acid, after which it is heated in the 
autoclave for thirty minutes at a pressure of 15 pounds, and is filtered 
through filter paper. The clear brown fluid is titrated again, and the 
reaction adjusted to 0.5 per cent. acid. Glycerin is added to the 
strength of 4 per cent, and the broth is divided among 250 cc. bottles, 
placing about 50 cc. in each bottle. It is then sterilized by fractional 
sterilization in the Arnold sterilizer on three successive days. 

After the bacilli have grown in the potato broth for six weeks, the 
broth is carefully filtered through several layers of filter paper, using 
suitable care to prevent contamination of the filtrate. Preservatives 
are not employed, since they eventually render the filtrate anti- 
complementary. Petroff states that when growth has proceeded 
sufficiently to produce an antigen of proper strength, the color of the 
potato broth changes from dark brown to golden yellow. The filtered 
antigen is placed in small ampoules containing 2 cc. each. These are 
stored in the refrigerator. 



OTHER SERUM TESTS 161 

Antigen II is monovalent. Two hundred and fifty milligrams of 
dried and pulverized tubercle bacilli are extracted with 50 cc. of 1 per 
cent, aqueous solution sodium hydrate at 38° C. for five to ten days, 
when the extract is carefully neutralized with decinormal hydrochloric 
acid. Petroff found that the antigen was quite stable. 

Antigen III. — Two hundred and fifty milligrams of dried and pul- 
verized tubercle bacilli are extracted with 50 cc. of Merck's reagent 
methyl alcohol at 38° C. for five to ten days, when the bottle is corked 
tightly and set away in a dark place at room temperature. At the 
end of several weeks' time, the supernatant clear fluid is pipetted off. 
The antigen is diluted with normal salt before use. 

After careful titration of the complement and amboceptor, Petroff 
recommends the use of the antigen in an amount equal to one-fourth 
of its anticomplementary unit. He uses an antisheep hemolytic 
system and allows the incubation of antigen, human serum and com- 
plement to proceed for one and a half hours. Presence of native anti- 
sheep amboceptor is noted and allowance is made. 

Wilson and von Wedel' s Method.— These workers employed the 
reagents in one-tenth the original Wassermann quantities. The 
antigens are prepared either from a number of stock cultures of human 
tubercle bacillus or from a strain used for the production of tuberculin, 
grown in glycerin broth for from three weeks to three months and 
then killed by heating in the Arnold sterilizer for one hour. The 
culture is then filtered through filter paper. The filtrate is discarded 
and the residue placed in absolute alcohol in the proportion of one 
volume of residue to ten volumes of alcohol. The mixture should be 
shaken thoroughly by hand and placed in the ice-box for two weeks 
when it is filtered through paper. The filtrate is discarded and the 
residue remaining, on the filter paper washed with absolute alcohol into 
a centrifuge tube. After centrifugalization the alcohol is removed and 
ether is added to the sediment, which is again centrifugalized. The 
ether is discarded and the centrifuge tube, plugged lightly with cotton, 
is allowed to stand at room temperature over night so that the residual 
ether may evaporate. One gm. of powder is emulsified in a large 
mortar with 200 cc. of normal saline and the emulsion is heated for one 
hour at 80° C. After standardization, it should be diluted so that the 
test-dose contains at least 2 antigenic units, and one-fourth or less of 
the anticomplementary dose. The unit is determined by titrating 
various amounts of antigen with 0.01 cc. of known positive tuberculous 
serum. The standard dilution of the two antigens employed by Wilson 
was 1 : 50. 

Von Wedel has shown that some sera from active tuberculous cases 
gave negative results when the test was made on the first day after 
bleeding but reacted positively when the same specimens were kept in 
the ice-box for a week and were tested later. Sera from known nega- 
tive cases gave negative results when so tested. Von Wedel also 
recommends carrying out the test with the quantity of serum usually 
11 



162 EXAMINATION OF THE BLOOD 

employed in the Wasserniann reaction (0.01 cc. for the system as modi- 
fied, 0.02 cc., and 0.04 cc, with controls for each containing twice the 
quantity). Of course the result is considered only when the control 
tube shows complete hemolysis. 

Wilson has shown that the sera obtained from different guinea-pigs 
varies greatly in its fixability by definite tuberculous serum, and that 
complement which is quite active in the hemolytic system may not be 
efficient for tuberculosis complement-fixation. Therefore she recom- 
mends testing the serum from each guinea-pig for its fixability with 
tuberculous antigen and known tuberculous serum before use in 
diagnostic tests. 

Clinical Value of the Complement-fixation Test for Tuberculosis.— 
At present there is no general agreement as to the technic and there 
is a great disparity in the results obtained by different workers, the 
most confusing feature being that no two workers have obtained similar 
results in using the same methods. The reaction is still in the experi- 
mental stage, and much work will have to be done to develop a technic- 
ally reliable method before the results of observations can be used as a 
basis for interpreting the significance of future findings. However, 
it appears that even with the present methods, a definitely positive 
reaction added to suggestive clinical findings assists in establishing a 
positive diagnosis, that a negative result does not exclude tuberculosis; 
and that positive results are more frequent with active than with 
inactive cases. 

From Petroff's method, Brown and Petroff have drawn the following 
conclusions : 

Far advanced cases react positively more frequently than do the 
moderately advanced, and the moderately advanced than the incipient. 
All patients with tubercle bacilli do not react positively (about 10 per 
cent, are negative). The complement-fixation test and the intra- 
dermic tuberculin test do not run a parallel course, the latter being far 
more persistent. A positive fixation text suggests observation of the 
patient for a time. The complement-fixation test is of more value to 
the clinician when negative than when positive in the determination 
of what patients need treatment. 

Von Wedel states that his results indicate that 100 per cent, of non- 
tubercular cases will give absolutely negative results; that nearly 100 
per cent, of primary and active cases will give positive results with the 
exception of the dying cases, and that about 25 per cent, of the partially 
inactive and the active cases will give only weakly positive results. 

CHEMICAL EXAMINATION OF THE BLOOD. 

Introductory.— Among its many functions, blood serves as a medium 
which carries food substances from the gastro-intestinal tract to the 
tissues and carries waste products from the tissues to the different 
organs by which they are secreted from the body. It is evident that 



CHEMICAL EXAMINATION OF THE BLOOD 



163 



the chemical examination of the blood would yield information of 
diagnostic value; much more, indeed, than may be obtained by the 
mere analysis of any of the excretions alone. The study of the blood 
gives us a very accurate idea as to the increase of certain unconsumed 
food substances, such as sugar, and the accumulation of undue amounts 
of waste materials, so that an insight may be had into the actual excre- 
tory capacity of the organism. The substances for which the present 
methods are particularly adapted are: blood sugar, the non-protein 
nitrogen of the blood, blood urea, blood creatinine, uric acid, and blood 
chlorides. 





Fig. 48 



Fig. 49 



Figs. 48 and 49. — Duboscq colorimeter. 



The development of microchemical colorimetric methods by Folin, 
by Benedict, and by Myers has placed within reach of the clinical 
laboratory quantitative chemical methods for the determination of 
many of the constituents of the blood. These methods have been 
reduced to sufficiently simple terms so that they may be employed by 
any one who has been trained in quantitative analysis. The chief 
difficulties are those attendant upon securing chemically pure reagents 
and upon making up the standard solutions with extreme accuracy. 
Once they have been surmounted, the actual performance of the 



164 



EXAMINATION OF THE BLOOD 



determinations need be no more difficult than the determination of the 
amount of chlorides in the urine, and will certainly reveal information 
of great value. 

The most costly piece of apparatus will be the colorimeter. The 
high price of the Duboscq instrument (Figs. 48 and 49) probably 
deterred the more prompt general employment of colorimetric methods. 
While this apparatus is excellent it is fortunately not the only one now 
available on the market. Kober has devised a very good apparatus 




50. — Kober colorimct 



Bock-Benedict colorimeter. 



which is made in this country, is somewhat less expensive than the 
Duboscq instrument and has certain points of mechanical superiority 
(Fig. 50). Bock and Benedict have brought out a colorimeter (Fig. 51) 
which some workers prefer to any of the larger models, feeling that when 
the colors are properly matched the two halves of the optical field blend 
more perfectly than with other models. Like the Duboscq and the 
Kober, it employs the plunger principle. It is extremely simple, light, 
and even less expensive than the Kober. (Manufactured by the 
Klett Mfg. Co., Inc., New York City). It is the one which the writer 



CHEMICAL EXAMINATION OF THE BLOOD 



165 



employs for routine work because of the superior matching of the color 
fields. Myers suggests a very inexpensive outfit, an adaptation of the 
principle used in the hemoglobinometers of the Gower type (Figs. 52 
and 53) in which a given quantity of standard solution is placed in 
one tube and the unknown is placed in the other, a suitable diluent 
being added to the unknown until the colors in the contents of the two 
tubes have the same depth of color. 

In the Duboscq, Kober, or Bock-Benedict instruments, the cups are 
placed in position and the colorimeter is set on a bench in front of a 
good light, preferably from the north. The mirrors are than adjusted 
so that both halves of the visual field are illuminated equally. The 
standard solution is placed in the left hand cup. In the first two 
forms, the cup must be brought to the zero mark to make certain 




W 




Fig. 52 Fig. 53 

Figs. 52 and 53. — Myers' colorimeter. 

that no air-bubbles are left in the cup to interfere with the passage of 
the light rays. With the cup raised as far as possible, the position of 
the zero mark of the vernier scale with regard to the other scale should 
be noticed. If they do not correspond, the vernier should be adjusted 
until the two zero marks are at exactly the same level. This may be 
done readily with the Kober, but is impossible with the Duboscq, where 
the necessary correction must be noted and allowance made in the 
readings. The cup containing the standard is then lowered into the 
desired position, usually so that the reading is at ten, fifteen, or twenty 
mm. and the right hand cup may be filled with the unknown. This is 
manipulated as was the other cup, raising it to the zero mark to expel 
the air, and correcting for the zero mark in the same way. In using 
the Bock-Benedict instrument, the cup for the standard requires no 



166 EXAMINATION OF THE BLOOD 

adjustment after filling; the cup containing the unknown is filled as 
with the other instruments. 

When the right hand cup has been filled with the unknown and 
adjusted properly, it is lowered gradually by the observer while he 
keeps his eye at the ocular of the instrument. The depth of color in 
one side of the optical field will remain stationary, of course, while 
that in the other grows increasingly deeper. When the two halves 
match, the reading is taken. It is desirable to take three or four 
readings with the same unknown and then average the results. 

Folin and Denis emphasize certain details in the use of the colori- 
meter. The zero points should be properly adjusted. The optical 
parts of the instrument should be thoroughly freed from dust so that 
hardly a single black speck is visible in either field. Care should be 
taken that the instrument is optically correct and that the two fields 
are equally lighted. The instrument should be used in a comfortable 
position and in a suitable light. Folin recommends putting the 
instrument on a stool about the height of an ordinary chair in the 
middle of the laboratory several feet from the window, and sitting at 
the side of the instrument instead of directly behind it facing the scale. 
In reading an unknown, both cups should be filled with the standard 
which should be read against itself to become familiar with the appear- 
ance of the standard solution. The standard in the right hand cup is 
then replaced with the unknown. Folin recommends making only 
one very careful reading of the unknown. 

The principle of the colorimeter is that that quantity of the substance 
in solution may be measured by the depth of color change produced. 
The depth of color change is then directly proportional to the strength 
of the solution and inversely proportional to the height of the column 
in the colorimeter cup. The equation which is used in calculating the 
results of colorimeteric determination is: 

RS : RX = CX : CS, 

in which RS is the reading of the standard, RX, the reading of the 
unknown, CX represents the concentration of the unknown and CS is 
the concentration of the standard. The reading of the standard and 
the concentration of the standard are known factors; when the reading 
of the unknown is ascertained, these three factors may be substituted 
in the above equation, and the concentration of the unknown deter- 
mined. With most of the methods given in this manual which employ 
colorimetric determination, a short formula will be given. 1 

Routine Procedure.— The tests which will be employed depend upon 
the nature of the case. In diabetes, the determinations which would 
be desired would include at least a determination of the blood sugar 
and of the C0 2 combining power according to Van Slyke's method. 

1 A list of apparatus required for chemical examination of the blood will be found 
in the appendix. The apparatus required for the tests for acidosis is specified with the 
descriptions of the tests. 



CHEMICAL EXAMINATION OF THE BLOOD 167 

These represent minimum requirements. Blood examination must be 
accompanied by a careful examination of the urine, with especial 
reference to the amount excreted in twenty-four hours, the amount of 
sugar in a twenty-four-hour specimen, the presence of the acetone 
bodies, and also the presence of albumin and the microscopic exami- 
nation. To these methods of examination may be added Sellards' 
bicarbonate tolerance test, and in indicated cases, the determination of 
the carbon-dioxide tension of the alveolar air. The determination of 
the diastatic activity of the blood may be helpful. 

With nephritis, the determinations of especial value include the 
estimation of blood urea, creatinine, and uric acid, and the determina- 
tion of the C0 2 combining power. When carrying out these deter- 
minations, the determination of blood sugar involves very little addi- 
tional labor and often contributes data of value. The determination 
of non-protein nitrogen adds little data of clinical usefulness when 
the blood urea has been determined, and imposes much labor. The 
work upon the blood should be supplemented by a careful study of the 
urine, particularly with reference to the presence and amount of 
albumin in a twenty-four-hour specimen, the amount voided in twenty- 
four hours, the microscopic study of a fresh specimen, the response 
to the Mosenthal diet, and the excretion of phenolsulphonephthalein. 1 

In gout the determination of urea, creatinine and especially of uric 
acid should be made. For the normal values and the findings in 
various diseases, the table on page 168. 

Obtaining Specimen of Blood.— The specimen should be obtained 
twelve to sixteen hours after a meal, preferably in the morning before 
breakfast, after a night during which no food has been taken. The 
specimen should be drawn directly from the vein into a tube into 
which has been placed powdered potassium oxalate. Twenty milli- 
grams of potassium oxalate is sufficient for 10 cc. of blood. An excess 
should be avoided. If a syringe is used the oxalate may be placed in 
the barrel of the syringe. Since other tests upon the blood may be 
performed at the same time, it is desirable to obtain at least 10 or 15 cc. 
of blood. For obtaining the specimen, a McRae needle or one of the 
type suggested by the writer, may be used. The technic for veni- 
puncture has been described on page 126. 

Choice of Methods.— For chemical determinations of sugar and the 
nitrogen derivatives, two methods have been given, those devised by 
Folin and Wu, and those adapted by Myers and his co-workers. The 
choice is largely a matter of individual preference. 

Folin and Wu employ tungstic acid as a protein precipitant. The 
technic for this preliminary step, which is common to all their methods, 
is given with the description of the determination of blood sugar, and 
to save needless repetition, is not repeated in describing the following 
methods devised by the same authors. When a complete examina- 

1 An excellent discussion of the chemical changes in the blood in disease is given 
by V. C. Myers in the Jour. Lab. and Clin. Med., 1920, v, 343, et. seq. 



168 



EXAMINATION OF THE BLOOD 



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DETERMINATION OF BLOOD SUGAR 169 

tion is to be made, an adequate quantity of blood is treated with 
tungstic acid and portions of the filtrate used for the different deter- 
minations. 

The same rule has been followed in describing Myers's set of tests, 
though it should be noted that his method for uric acid is an adapta- 
tion of Folin and Wu's and calls for precipitation of the blood by 
tungstic acid rather than by picric acid, as do his other tests. 

General Method of Procedure.— If Folin and Wu's methods are to be 
used, precipitate 10 cc. of blood with tungstic acid (see page 172) and 
use portions of the filtrate for determinations of sugar, creatinine, 
urea, non-protein nitrogen, and uric acid, or as many of these tests 
as may be required. If Myers's methods are to be followed, precipi- 
tate 5 cc. of blood with picric acid (see below) for sugar and creati- 
nine determinations, precipitate another 5 cc. portion with tungstic 
acid (see page 194) for uric-acid determination, use whole blood for 
the estimation of urea, and treat a portion with trichloracetic acid 
for the determination of non-protein nitrogen. While this may seem 
somewhat more involved, the author has not found the latter set of 
tests required an appreciably greater number of operations than the 
former. 

DETERMINATION OF BLOOD SUGAR. 

Method of Myers and Bailey (Modified from that of Benedict and 
Lewis).— Principle.— The blood proteins are precipitated by picric 
acid in saturated solution. The addition of sodium carbonate to 
picric acid solution results in the formation of sodium picrate, which 
in the presence of heat, is reduced by the sugar in the filtrate with 
a color change which may. be determined by colorimeteric methods, 
using as a standard a solution of glucose of known strength. 

Reagents Required.— 1. Standard solution of glucose, 0.02 per cent, in 
a saturated solution of picric acid. 

2. Saturated solution of sodium carbonate (about 22 per cent.). 

3. Picric acid, pure, crystals. 

For method of testing purity of picric acid and for purifying it, 
see page 442. 

Procedure.— Should only the blood sugar determination be desired, 
2 cc. of blood are laked by the addition of 8 cc. of water. If, however, 
one desires to determine the amount of creatinine at the same time, 5 cc. 
of blood may be laked by the addition of 20 cc. of water. This laking 
is accomplished conveniently in a 50 cc. round-bottomed centrifuge 
tube. Dry chemically pure picric acid is added to the point of satu- 
ration. The picric acid is stirred thoroughly with a glass rod and is 
added bit by bit until a few undissolved crystals are seen at the bottom 
of the tube. After the picric acid has been thoroughly mixed with 
the laked blood, the mixture is allowed to stand about five minutes 
when separation may be hastened by centrifugalization, and the 
supernatant fluid is filtered through paper. If a heavy centrifuge be 



170 



EXAMINATION OF THE BLOOD 



not available, it may, of course, be filtered without centrifugalization. 
The precipitate is composed largely of protein, sugar, of course, being 





m 




Fig. 54 — Types of glassware used in blood chemistry, a, Folin pipette; b, volu- 
metric pipette with one mark only; c, 10 cc. pipette, graduated in 0.1 cc. ; d, Ostwald 
pipette; e, 1 cc. pipette, graduated in 0.1 cc; /, 50 cc. burette, graduated in 0.1 cc; 
g, graduated 15 cc. centrifuge tube; h, graduated 50 cc. centrifuge tube; i, 10 cc. cylin- 
drical graduate; /, volumetric flasks (various capacities); k, Folin ignition tube; I, 
Myers eugar tube; m, Folin sugar tube. 



in solution. Three cubic centimeters of the filtrate are placed in a 
special Myers' sugar tube (Fig. 54, /). At the same time one should 
set up as a control a tube of the same type containing 3 cc. of the 



DETERMINATION OF BLOOD SUGAR 171 

standard 0.02 per cent, glucose solution made in saturated picric acid 
solution. To the tube containing the unknown and also the control 
tube there should be added 1 cc. of sodium carbonate solution. The 
two tubes are then placed in a beaker of water, which is boiled for at 
least ten minutes. At the end of this time the fluid in the tube con- 
taining the standard solution is brought to the 10 cc. mark with 
distilled water. The fluid in the tube containing the unknown is 
brought to the 10, 15, 20, or even 25 cc. mark as may be required to 
bring the colors to about the same depth. When the colors have been 
matched roughly in this way, the standard solution is brought to 
the left hand cup of the colorimeter and the fluid from the tube con- 
taining the unknown is placed in the right hand cup of the colorimeter. 
The standard solution is placed at the 10 mm. mark and the tube 
containing the unknown is moved back and forth until the two fields 
match when the reading of the unknown is taken. 
Resume of Method.— 

1. To 2 cc. of oxalated blood add 8 cc. of distilled H 2 0. 

2. Add dry picric acid to saturation, mixing thoroughly. 

3. Let stand five minutes, then centrifugalize. 

4. Filter and employ filtrate as shown below (tube with unknown). 

5. Place 3 cc. standard 0.02 per cent, glucose solution in Myers tube. 

TUBE WITH STANDARD. TUBE WITH UNKNOWN. 

6. Put in 3 cc. of standard glucose Put in 3 cc. of filtrate. 

solution. 

7. Add 1 cc. sat. sol. Na 2 C0 3 . Add 1 cc. sat. sol. Na 2 C0 3 . 

8. Stand in beaker of boiling Stand in beaker of boiling 

water 10 minutes. water 10 minutes. 

9. Add distilled H 2 to 10 cc. Add distilled water to 10, 15, 

20 cc. mark, as needed to ap- 
proximate color of standard. 

10. Compare tubes in colorimeter. 

Calculation. — When the procedure has been carried out as outlined 

above, the following formula may be used for computation : 

D X ~- = the blood sugar in per cent. 
Jbi 

Folin and Wu's Method for Sugar Determination.— Folin has criticized 
the picrate methods stating that in his hands they have given results 
which are higher than with his method. He suggests that the picrate 
methods may be subject to sources of error similar to those encountered 
in his original method for blood creatinine. The color developed with 
this method is far more intense than that developed by the alkaline 
picrate so that a small fraction of a milligram of dextrose is all that is 
required for a determination. While sensitive to traces of dextrose, 
the copper solution is not affected by creatinine or uric acid in quanti- 
ties corresponding to 50 mg. per 100 cc. of blood. 



172 EXAMINATION OF THE BLOOD 

Principle.— The proteins are precipitated from the blood by tungstic 
acid. The protein-free filtrate is allowed to reduce a weakly alkaline 
solution -of copper tartrate, with the aid of heat. Folin and Wu's 
molybdate phosphate reagent is added, which forms an intense blue 
color in the presence of cuprous oxide in acid solution. The amount 
of dextrose may be estimated colorimetrically by comparison with a 
standard containing a known amount of dextrose which has been sub- 
jected to similar treatment. 

Reagents Required.— 1. Stock and Standard Sugar Solutions.— The 
stock sugar solution is prepared by dissolving 1 gm. pure anhydrous 
dextrose in water and diluting to 100 cc. 

When pure dextrose is not available, a standard solution may be made 
from invert sugar prepared from cane sugar. Exactly 1 gm. cane sygar 
is placed in a 100 cc. volumetric flask (Fig. 54, j) with 20 cc. of normal 
hydrochloric acid. The mixture is allowed to stand at room tempera- 
ture overnight. This step may be carried out in ten minutes by rotat- 
ing the flask and its contents continuously in a water-bath at 70° C. 
The acid is neutralized by adding 1.68 gm. sodium bicarbonate and 
about 0.2 gm. sodium acetate. It is necessary to shake for a few 
minutes to remove the carbonic acid. The flask is filled to the 100 cc. 
mark with distilled water. An additional 5 cc. of water is added, since 
1 gm. cane sugar yields 1.05 gm. invert sugar. The contents of the 
flask are mixed thoroughly and poured into a bottle with a few drops of 
xylene or toluene. The stock solution, made either from dextrose or 
invert sugar, keeps indefinitely. 

Two standard solutions are prepared from the stock solution, one 
by diluting 5 cc. of the stock solution to 500 cc. with distilled water and 
the other by diluting 5 cc. of the stock solution to 250 cc. Two cc. of 
the first solution contains 0.2 mg. dextrose, and 2 cc. of the second con- 
tains 0.4 mg. The standard solution may be kept with xylene or 
toluene, but should not be depended upon for more than a month. 

2. Alkaline Copper Tartrate Solution.— Forty gm. anhydrous sodium 
carbonate are dissolved in 400 cc. distilled water and transferred to 
a liter volumetric flask. In this solution are dissolved first 7.5 gm. 
tartaric acid and then 4.5 gm. crystallized copper sulphate. The 
volume is made up to 1 liter after thorough mixing. Should a sediment 
appear as the result of impure chemicals, the clear supernatant fluid 
may be removed with a siphon. 

3. Folin and Wu's Molybdate-phosphate Reagent.— 1\\ a liter beaker 
is placed 35 gm. molybdic acid, 5 gm. sodium tungstate, 200 cc. of 
10 per cent, sodium hydrate and 200 cc. of water. This is boiled 
vigorously for twenty to forty minutes to remove nearly all the 
ammonia which the molybdic acid contains as an impurity. The 
solution is cooled and diluted to 350 cc. when 125 cc. concentrated 
phosphoric acid (85 per cent.) are added and the volume brought to 
500 cc. with distilled water. 



DETERMINATION OF BLOOD SUGAR 173 

4. Sodium Tungstate.—A 10 per cent, solution in distilled water. 
Folin and Wu have found a number of brands satisfactory.. (It may be 
obtained from the American Vanadium Corporation, Primos, Pa., 
who are successors to the Primos Chemical Co., mentioned by Folin 
and Wu as having made this chemical for them, and market a brand 
known as "Dr. Folin's quality.") The impurity which may be bother- 
some is an excess of carbonates. This may be tested for as follows 
(Folin and Wu) : A drop of phenolphthalein is added to 10 cc. of the 
10 per cent, sodium tungstate solution, and y^ HC1 is added from a 
burette until the color fades. Each cc. of the decinormal acid repre- 
sents 1.06 per cent, of sodium carbonate. With a satisfactory pre- 
paration, the amount of acid required for neutralization should not 
exceed 0.4 cc. 

5. Two-thirds Normal Sulphuric Acid Solution. 

Procedure.— Blood is obtained by venipuncture and oxalated as 
has been directly previously (see page 167). When only a sugar 
examination is desired, start with 2 cc. of blood, but if a complete 
set of tests be required, 10 cc. of blood should be used and the reagents 
added in corresponding proportions. 

The blood is placed in a flask having a capacity fifteen to twenty 
times that of the volume taken. To it should be added in turn, 
seven times its volume of water, 1 volume of 10 per cent, sodium 
tungstate, and 1 volume of two-thirds normal sulphuric acid; the 
final dilution of blood is 1 to 10. The contents of the flask should 
be mixed after the addition of each reagent and shaken constantly 
during the addition of the sulphuric acid. Then the mouth of the flask 
is closed with a rubber stopper and the flask is given a few vigorous 
shakes. When conditions are right, scarcely an air-bubble is formed. 

When properly coagulated, the color of the coagulum changes from 
pink to dark brown. When the change does not occur, the fault 
lies in the addition of too much oxalate. This fault may be remedied 
by adding 5 per cent, sulphuric acid, drop by drop, shaking vigorously 
after each addition, and allowing the flask to stand for a few minutes 
before adding more, until coagulation is complete. The mixture 
should be poured on a filter large enough to hold the entire contents 
of the flask, when the funnel is covered with a watch glass to prevent 
evaporation. If the filtrate be cloudy, the first few cubic centimeters 
should be returned to the funnel. 

A water-bath is heated to boiling. Into one of the special Folin 
sugar tubes (Fig. 54) graduated to 25 cc. is placed 2 cc. of the blood 
filtrate. Into each of 2 similar tubes are placed 2 cc. of the standard 
sugar solutions, containing respectively 0.2 and 0.4 mg. of dextrose. 
To each tube is added 2 cc. alkaline copper tartrate solution. The 
surface of the mixture should reach the constricted part of the bulb. 
If the bulb be too large, 0.5 cc. or less of diluted (1:1) alkaline copper 
tartrate solution may be added. If this does not suffice to bring the 
contents to the narrow part, or if the tube is so small that the fluid 



174 



EXAMINATION OF THE BLOOD 



does not come up to the neck, the tube should be discarded. The 
tubes are placed in boiling water and heated for six minutes. They 
are removed and cooled in a cold water-bath for two to three minutes 
without shaking, when 2 cc. of molybdate-phosphate solution are 
added to each tube. After the cuprous oxide has dissolved, the 
contents of all tubes are diluted to the 25 cc. mark. Rubber stoppers 
are inserted and the solutions are mixed. The unknown solution is 
compared colorimetrically with that standard whose color intensity 
most nearly approximates its own. 
Resume of Method. — 

1 . Place 1 volume of oxalated blood in an Erlenmeyer flask. 

2. Add (a) 7 volumes of distilled water. 

(b) 1 volume of 10 per cent, sodium tungsate. 

(c) 1 volume of two-third normal sulphuric acid. 
Rotate flask constantly while making additions. 

3. Filter when coagulation is complete. Employ portion of filtrate 

as shown below. 
(Steps 1 to 3 inclusive are the same as those employed for obtaining 
protein-free filtrate for other determinations by Folin and Wu's 
methods. If these be desired, take a larger quantity of blood 
and increase quantities of reagents in 2 accordingly.) 



TUBE WITH FIRST 
STANDARD. 

4. 2 cc. standard sugar 

solution (contains 
0.2 mg. dextrose). 

5. Add 2 cc. alkaline 

copper tartrate 
solution. 

6. Place in boiling 

water 6 minutes. 

7. Remove and place in 

cold water 2-3 
minutes. 

8. Add 2 cc. molyb- 

date-phosphate 
solution. 

9. Allow cuprous oxide 

to dissolve. 
10. Dilute to 25 cc. mark 



TUBE WITH SECOND 
STANDARD. 

2 cc. standard sugar 
solution (contains 
0.4 mg. dextrose). 

Add 2 cc. alkaline 
copper tartrate 
solution. 

Place in boiling 
water 6 minutes. 

Remove and place in 
cold water 2-3 
minutes. 

Add 2 cc. molyb- 
date-phosphate 
solution. 

Allow cuprous oxide 
to dissolve. 

Dilute to 25 cc. mark 
with water. 



with water. 
Compare contents in colorimeter. 
Calculation.— The calculation may be made 
representing the reading of the cup containing 
that containing the unknown. 

s x 



Mg.'of sugar per 100 cc. of blood = 



TUBE WITH 
UNKNOWN. 

2 cc. protein-free fil- 
trate. 

Add 2 cc. alkaline 
copper tartrate 
solution. 

Place in boiling 
water 6 minutes. 

Remove and place in 
cold water 2-3 
minutes. 

Add 2 cc. molyb- 
date-phosphate 
solution. 

Allow cuprous oxide 
to dissolve. 

Dilute to 25 cc. mark 
with water. 

by this formula, S 
the standard and R 

100 



DETERMINATION OF BLOOD SUGAR 175 

Findings.— In normal cases the findings run between 0.09 and 0.12 
per cent., according to the figures of Lewis and Benedict, and Myers 
and Bailey. Myers and Bailey, however, state that there are a number 
of hospital cases which show blood sugars as high as 0.14 per cent. 
In renal diabetes there is no increase in the amount of sugar in the 
blood. An increased amount of blood sugar has been reported in fatal 
cases of mercuric bichloride poisoning with nephritis; in a case of 
chronic interstitial nephritis; and in numerous cases of chronic paren- 
chymatous nephritis. 

Diastatic Activity of the Blood.— While it has been recognized since 
the time of Magendie that the blood possessed active diastatic proper- 
ties, there had been no conclusive work done in regard to this property 
in human beings subject to diabetes till the work of Myers and Killian. 
From their study they conclude that the diastatic activity of the blood 
is increased in cases of diabetes and also in cases of nephritis. The 
method is simple. 

Reagents Required. — Those specified for blood and sugar determi- 
nations with the addition of 1 per cent, solution of soluble starch in 
water. The sugar content of this should be determined. 

Procedure.— Oxalated blood is obtained as previously directed. 
Two tubes are prepared, each containing 2 cc. of blood. One tube 
serves as a control. This should be marked C. The other tube is 
for the purpose of determination. This should be marked A. To the 
tube marked C should be added 8 cc. of water, while to the tube marked 
A are added 7 cc. of water. These tubes are placed in an incubator or 
water-bath at 40° C. for ten minutes, at the end of which time 1 cc. of 
a 1 per cent, solution of soluble starch is added to tube A. The tubes 
are again placed in the incubator or water-bath at 40° C. for fifteen 
minutes, at the end of which time the sugar is determined from both 
tubes according to the method previously given of Myers and Bailey. 
The amount of sugar in the starch solution should have been determined 
and should be expressed in milligrams. 

Resume of Method.— 

TUBE A. TUBE C. 

1. 2 cc. of oxalated blood. 2 cc. of oxalated blood. 

2. Add 7 cc. of distilled water. Add 8 cc. of distilled water. 

3. Leave in water-bath at 40° C. Leave in water-bath at 40° C. 

for ten minutes. for ten minutes. 

4. Add 1 cc. of 1 per cent, solution 

soluble starch. 

5. Replace in water-bath at 40° C. Replace in water-bath at 40° C. 

and leave fifteen minutes. and leave fifteen minutes. 

6. Determine the amount of sugar in each specimen by method given 

previously (see page 169). 

Calculation.— After the amount of sugar in tubes A and C has been 
determined in percentages according to the Myers and Bailey method, 
it should be translated to terms of mg. per cc. The number of mg. 



176 EXAMINATION OF THE BLOOD 

in 2 cc. of the blood before starch has been added, as determined by 
tube C, should be subtracted from the amount of sugar in the specimen 
to which starch has been added as determined by tube A. From this 
should be subtracted the corrections for the amount of free sugar in 
the starch solution. The remainder will represent mg. of the sugar 
which has been formed from the starch by the diastatic activity of 
2 cc. of blood. This may be transferred to terms of percentage since 
the amount of the starch, being a 1 per cent, solution, contained 10 
mg. of starch. For example: If 2 cc. showed that there was 0.22 
per cent, of sugar in the blood, 2 cc. of blood would contain 4.4 mg. 
If tube A showed that the conversion of starch by the diastase had 
brought the total of blood sugar plus converted starch to 0.4 per cent, 
sugar, then in 2 cc. there would be 8 mg. of sugar. If the starch 
solution contained 0.6 mg. of sugar (and it should not exceed this figure) 
we would subtract 4.4 and 0.6 from 8 leaving a remainder of 3 mg. 
which represents the diastatic activity of 2 cc. of blood. In other 
words, the blood was able to convert 30 per cent, of the amount of 
starch present. 

Significance of Findings— By this method Myers and Killian found 
that the diastatic activity of the blood in normal subjects ranged from 
15 to 20. In cases of diabetes they found the figure ranged from 25 to 
75, and in cases of nephritis, from 20 to 50. They thought that the 
results suggested that increased diastatic activity in both diabetes and 
nephritis may be an important factor in the production of hypergly- 
cemia; that a fall in the blood diastase would appear to afford a more 
reliable guide to the efficacy of treatment in diabetes than either the 
blood sugar or the urinary sugar; and that an increase in the blood 
diastase may constitute a very early sign of impending diabetes. 

Determination of Sugar Tolerance.— Hammann and Hirschman 
proposed utilizing the determination of alimentary hyperglycemia 
and glycosuria as a clinical method for sugar tolerance. They found 
that the maximum concentration reached from 0.1 per cent, to 0.13 
per cent, after giving 100 grams of glucose, and that the high point 
was attained from twenty minutes to one and a half hours. 

Their method was essentially as follows: After a night's fast, the 
patient is given 100 gin. of glucose dissolved in 300 cc. of water, 
flavored with orange or lemon juice. Blood is taken by venipuncture 
before giving the glucose, one-half hour later, one hour later, and 
finally two hours later, and the blood sugar is determined by one 
of the preceding methods. At the same time-intervals, specimens of 
urine are obtained and examined for sugar by Benedict's qualitative 
and quantitative "methods. 

Hammann and Hirschman found that patients with lowered car- 
bohydrate tolerance presented more pronounced and more prolonged 
hyperglycemia than was seen in normal individuals, the blood sugar 
usually "exceeding 0.2 per cent, and the reaction lasting from three 
to five hours. Increased carbohydrate tolerance was indicated by 
subnormal- reaction, the blood sugar showing only an insignificant 



BLOOD UREA 177 

rise. As far as could be determined from this alimentary test, the 
disturbance in glucose utilization was essentially the same in diabetes 
and in other conditions with low sugar tolerance, such as deranged 
thyroid and hypophysial function, and in nephritis. 

It was shown that the normal renal threshold for glucose was 
between 0.17 and 0.18 per cent., that is, that glucose appeared in 
the urine when the blood sugar reached this concentration. 

A normal threshold was found in patients with diminished carbo- 
hydrate tolerance, while some otherwise normal individuals have a 
low renal threshold. Although the threshold is usually normal in 
mild cases of diabetes, it may be lowered in severe case, this factor 
contributing to the severity of the condition. 

The test proves particularly valuable in the investigation of patients 
with slight or occasional glycosuria, since it decides whether the 
glycosuria be due to a metabolic disturbance, to a low renal threshold, 
or to a combination of both factors. A high renal threshold suggests 
a more serious disturbance of glucose utilization than would be indi- 
cated by a mild glycosuria. 

BLOOD UREA. 

Method of Myers (Modified from those of Marshall and of Van Slyke 
and Cullen).— Principle.— By the aid of the ferment, urease, the blood 
urea is converted to ammonium carbonate. This is broken up and the 
ammonia liberated by the action of a strong alkali (saturated solution 
of sodium carbonate). By means of a strong current of air, the 
ammonia is removed and neutralized by sulphuric acid. By Ness- 
lerizing the resultant solution of ammonium sulphate, the amount 
of nitrogen may be determined. 

Reagents Required. — 1. Urease, 5 per cent, solution in water, freshly 
prepared. The brand of urease sold by the Arlington Chemical Com- 
pany is preferable. It contains monobasic and dibasic phosphates in 
proper proportion to give a neutral solution when dissolved. 1 

2. Decinormal sulphuric acid. 

3. Sodium carbonate, saturated solution. 

4. Nessler's solution (Benedict-Bock formula). 
This is prepared as follows: 

Mercuric iodide 50 gm. 

Potassium iodide 35 " 

Sodium hydrate 50 " 

Distilled water 500 cc. 

1 Folin and Wu recently called attention to the fact that preparations of urease do 
not show constant keeping power and suggest the following method of preparation: 
Transfer to a 200 cc. flask about 3 gm. of permutit powder. Wash this by decanta- 
tion, once with 2 per cent, acetic acid, then twice with water. Add to the moist per- 
mutit in the flask 100 cc. of 30 per cent, alcohol (35 cc. of 95 per cent, alcohol mixed 
with 70 cc. of water). Then introduce 5 gm. jack bean meal (may be obtained from the 
Arlington Chemical Co.) and shake for 10 minutes. Filter and collect the filtrate in 
three or four different small clean bottles. Set one aside for immediate use; it will 
remain serviceable for at least 1 week at ordinary room temperature, if not exposed to 
direct sunlight. Put the others on ice where they will remain good for 3 to 5 weeks. 
12 



178 



EXAMINATION OF THE BLOOD 



The mercuric iodide is rubbed to a smooth paste in a mortar with 
a little water and is transferred to a 500 cc. measuring flask. The 
potassium iodide is ground to a powder in the same mortar and 
added to the iodide paste in the measuring flask, washing it with about 
200 cc. of water. The sodium hydrate is dissolved in about 250 cc. 
of water and, after cooling, is added with constant shaking to the solu- 
tion of mercuric iodide and potassium iodide in the measuring flask. 
The volume is brought to 500 cc. with distilled water. After stand- 
ing at room temperature until the yellowish-white precipitate, which 
may settle out at first, has dissolved, the supernatant clear fluid of 
siphoned off from the dark-brownish-red precipitate. 




Wash Bottle 
containing 
dilute i/ z S0 4 



Fig. 55. — Aerating apparatus. 



5. A standard solution of ammonium sulphate (containing 1 mg. is 
nitrogen to 5 cc. of solution). A standard solution to contain 1 mg. 
of nitrogen per 5 cc. of solution may be prepared by dissolving in a liter 
of distilled water 0.944 gm. of ammonium sulphate or 0.764 gm. of 
ammonium chloride, highest purity. Folin states that pure ammonium 
sulphate may be prepared by decomposing a high grade ammonium 
salt with sodium hydroxide and passing the ammonium gas which is 
liberated by this decomposition through pure sulphuric acid with the 
aid of an air current. The salt, which is redissolved in water, is again 
precipitated with alcohol, and is finally dried in a desiccator over 
sulphuric acid. 

Procedure.— Blood is obtained by venipuncture and oxalated as 
previously directed (see page 167). The aerating apparatus is set up 



BLOOD UREA 179 

(Fig. 55). It should be tested out before making the determination 
by connecting it and then turning on the air current. 

If all joints are air-tight, air should bubble through the dilute acid 
solution in wash bottle (W). In graduate B is placed 2 cc. of -jo 
H 2 S0 4 and 15 cc. of water. 

Into a test-tube is placed 2 cc. of the oxalated blood, and to it is 
added 1 cc. of the 5 per cent, urease solution. The test-tube is then 
placed in the water-bath at 50° C. for fifteen minutes, at the end of 
which time the test-tube is placed inside the graduate (A) after adding 
1 cc. of amyl alcohol to prevent bubbling. With the tube in place 
and the apparatus ready to connect, 4 to 5 cc. of saturated solution 
of sodium carbonate is run in with a pipette. The alkali facilitates 
breaking up of the ammonium carbonate to ammonia. Aeration is 
carried out by using a strong current of air. The apparatus should 
be set up as is shown in Fig. 55. The ammonia gas which is freed by 
aeration is drawn into the second graduate -(A). Aeration is con- 
tinued from thirty to forty-five minutes. 

The standard should be made up in a 100 cc. graduated cylinder 
in which are placed 5 cc. the standard ammonium sulphate solution 
(containing 1 mg. nitrogen), 20 cc. distilled water, 20 cc. 20 per cent. 
Nessler's solution, and water sufficient to bring the total volume up to 
100 cc. To the cylinder which contained the decinormal acid and 
into which the ammonia has been drawn by the air current is added 
enough 20 per cent. Nessler's solution to secure maximum color change 
(this may be 20 cc. or less) and water to make the intensity of the color 
of the standard and the unknown approximately the same (usually 
stopping at 30, 50, or 100 cc, for the sake of convenience in compu- 
tation). 

The left hand cup of the colorimeter is then charged with a portion 
of the standard solution and the right hand cup is charged with a 
portion of the unknown. The left hand cup is set at 10 mm. and the 
right hand cup is moved up and down until the color in the two halves 
of the colorimeter field matches when the reading is taken. 

Resume of Method. — 

1. Place 2 cc. of oxalated blood in test-tube. 

2. Add 1 cc. of 5 per cent, urease solution. 

3. Leave in water-bath at 50° C. for fifteen minutes. 

4. Add 1 cc. of amyl alcohol. 

5. Place test-tube inside cylinder A of aerating outfit. 

6. In receiving cylinder B, place 2 cc. of yir H 2 S0 4 and 15 cc. dis- 

tilled water. 

7. Add 5 cc. of saturated solution of sodium carbonate to tube in 

cylinder A. 

8. Stopper cylinders A and B, connect up apparatus, and start air 

current. 

9. Allow aeration to proceed for from thirty to forty-five minutes. 
10. Take cylindrical graduate C of the same size as B and mix in it 

the standard solution. 



180 EXAMINATION OF THE BLOOD 

Treat the contents of the cylinders B and C as follows : 

cylinder b (contains the cylinder c (contains the 

unknown). standard). 

11. Contains substances placed in Place in cylinder 5 cc. standard 

it in step 6 plus the liberated ammonium sulphate solution 

ammonia. (contains 1 mg. nitrogen). 

12. Add 20 cc. distilled water. 

13. Add 20 per cent, dilution Ness- Add 20 cc. of 20 per cent, dilu- 

ler's solution to maximum tion of Nessler's solution, 

color. 

14. Dilute to 30, 50, or 100 cc. to Bring total volume to 100 cc. 

match roughly the color of C. with distilled water. 

15. Compare contents of cylinders in colorimeter. 

Calculation. — When the standard cup of the colorimeter is set at 
10 mm., divide one-half of the dilution of the unknown by the reading 
of the unknown and multiply the quotient by 10. This represents 
the mg. of urea nitrogen per 100 cc. of blood. The result is in terms 
of nitrogen. If it be desired to convert the urea nitrogen to terms of 
urea this may be done by multiplying by the factor 2.14. 

Method of Folin and Wu. — Principle.— The principle is essentially 
the same as in the foregoing method, except that protein is precipitated 
by tungstic acid. 

Reagents Required.— I. Sodium Tungstate (10 per cent.).— See page 
173. 

2. Two-thirds Normal Sulphuric Acid. 

3. Urease Solution.— See footnote, page 177. 

4. Pyrophosphate Solution .—Prepared by dissolving 140 gm. of 
sodium pyrophosphate (U. S. P.) in water, adding 20 gm. of glacial 
phosphoric acid, and bringing total volume to 1 liter with water. 

5. Standard Nitrogen Solution.— This may be prepared by dissolving 
0.2832 gm. of ammonium sulphate (highest purity) in distilled water 
and bringing volume to 1 liter with distilled water; 5 cc. of this will 
contain 0.3 mg. of nitrogen. 

6. Ticentieth-normal Hydrochloric Acid Solution. 

7. Saturated Solution of Borax. 

8. Paraffin Oil. 

9. Nesslers Solution (Folin and Wu's Formula).— A stock solution 
of mercuric potassium iodide is prepared by dissolving 150 gm. of 
potassium iodide in 100 cc. of warm water, adding 200 gm. of mercuric 
iodide, stirring until the latter is dissolved, and diluting to a volume 
of about 1 liter. This is allowed to settle when it is filtered and 
diluted to a final volume of 2 liters. It is advisable to make this in 
large volumes since a second sediment may form which requires a 
long time to settle. 

From this stock solution the final Nessler's solution is made as 
follows: A saturated solution of caustic soda is prepared and the 



BLOOD UREA 181 

clear supernatant fluid is decanted. This is diluted to a concentration 
of about 10 per cent. While the saturated solution contains about 
55 gm. of the alkali per 100 cc, it is wise to check the concentration 
by titration against a standard acid. Into a large bottle is intro- 
duced 3500 cc. of this 10 per cent, sodium hydrate solution with 750 
of the mercuric potassium iodide solution and 750 cc. of water, making 
the total volume of solution 5000 cc. 1 

Procedure.— Blood is obtained and oxalated as for other chemical 
procedures (see page 167). Protein is precipitated by sodium tung- 
state and sulphuric acid as described previously (see page 173). Five 
cc. of the filtrate (corresponding to 0.5 cc. of blood) are transferred to 
a clean dry Pyrex ignition tube with a capacity of about 75 cc. The 
tubes should be kept for this purpose alone and should be free from 
mercury or precipitated Nessler's solution. To the blood filtrate are 
added 2 drops of the pyrophosphate solution and 0.5 to 1 cc. of urease 
solution, when the tube is immersed for five minutes in a beaker of 
warm water, the temperature of which does not exceed 55° C. 

The ammonia may be distilled into 2 cc. of twentieth-normal hydro- 
chloric acid contained in a second test-tube, or it may be liberated and 
drawn by air current into a second tube containing 2 cc. of twentieth- 
normal hydrochloric acid. The method for arranging aspirating outfit 
for the latter method has been given in describing the preceding 
technic for determining urea (see page 179) . If aspiration be employed, 
add 1 or 2 cc. of 10 per cent, sodium hydrate to the hydrolyzed blood 
mixture just before aspirating. The calculation is the same in either 
case. 

Distillation may be performed with the aid of very simple appa- 
ratus. The tubes containing the treated specimen which is to be 
distilled and that containing the acid are both fitted with rubber 
stoppers, each pierced by a single hole. The stopper for the second 
or receiving test-tube should be cut with a fairly deep notch to permit 
the escape of steam and air. A piece of glass tubing is bent into a V 
form, so that the arms will be unequal. The shorter one is inserted 
through the stopper of the tube containing the specimen, the longer 
one through the stopper in the receiving tube. The end of the first 
arm should come just below the stopper while the tip of the second 
arm should dip into the acid in the receiving tube. No condensing 
apparatus is needed. 

1 On account of the difficulty of obtaining high-grade mercuric iodide, Folin and 
Wu offer the following method of preparing the double iodide solution: Place 150 
gm. of potassium iodide and 110 gm. of iodine in a 500 cc. Florence flask. Add 100 
cc. of water and an excess of metallic mercury (140 to 150 gm.). Shake the flask con- 
tinuously for ten to fifteen minutes until the dissolved iodine has nearly disappeared. 
When the red iodine solution has begun to pale visibly but while it is still red, cool 
the hot solution in running water while shaking until the red color of the iodine has 
been replaced by the greenish color of the double iodide. This usually requires about 
fifteen minutes. Decant the clear solution from the surplus mercury. Wash the 
mercury liberally with water, adding the washings to the solution. Dilute mixture 
of solution and washings to a volume of 2 liters. This preparation may be diluted 
with alkali exactly as the other solution of mercuric potassium iodide, but is usually 
clear enough for immediate dilution. The cost is less, and impurities are avoided. 



182 EXAMINATION OF THE BLOOD 

To the hydrolyzed blood filtrate is added a dry pebble, 2 cc. of 
saturated borax solution, and a drop or two of paraffin oil. The 
rubber stoppers are inserted firmly in both test-tubes, and the hydro- 
lyzed filtrate is boiled moderately fast over a microburner for four 
minutes. The size of the flame should not be cut down during this 
time nor the boiling should be so brisk that emission of steam from 
the receiving tube begins before the end of three minutes. At the 
end of four minutes the stopper is removed from the receiving tube 
and the end of the V-shaped delivery tube is held near the top of the 
receiving test-tube, while distillation is continued for one minute. 
The lower part of the delivery tube is rinsed off with a little water 
into the receiving tube. 

The distillate should be cooled with running water, when it is diluted 
to about 20 cc. Then 2.5 cc. of Nessler's solution are added and the 
volume is brought up to exactly 25 cc. The unknown is placed in 
the colorimeter and compared with a standard nitrogen solution. 
This is prepared by placing 0.3 mg. of nitrogen in a 100 cc. volumetric 
flask and adding 10 cc. of Nessler's solution and then diluted to 100 cc. 
Resume of Method— (Steps one to three inclusive are the same as 
those used for securing protein-free filtrate for blood sugar determina- 
tions, see page 173. Amount of blood used depends upon number of 
tests to be done.) 

1. Place one volume of oxalated blood in an Erlenmyer flask. 

2. Add (a) 7 volumes of distilled water. 

(6) 1 volume of 10 per cent, sodium tungstate. 
(c) 1 volume of two-thirds normal sulphuric acid. 
Rotate flask constantly while making additions. 

3. Filter when coagulation is complete. 

4. Place 5 cc. of protein-free filtrate in Pyrex ignition tube. 

5. Add (a) 2 drops of pyrophosphate solution. 

(b) 0.5 to 1 cc. of urease solution. 

6. Immerse tube in warm water for five minutes. 

7. Arrange distilling apparatus, placing 2 cc. ^r HC1 in receiving 

tube. 

8. Add to blood (a) a dry pebble. 

(b) 2 cc. of saturated borax solution. 

(c) a drop of paraffin oil. 

9. Insert stoppers in both tubes of distilling apparatus, and boil 

blood mixture over microburner for five minutes as directed. 

10. Rinse tip of delivery tube into receiving tube with a little distilled 

water. 

11. Cool distillate and add about 20 cc. of water. 

12. To distillate, add 

(a | 2.5 cc. of Nessler's solution (Folin and Wu's formula). 
{b) Distilled water to make volume of 25 cc. 

13. Prepare standard as follows: 

(a) 5 cc. of standard nitrogen solution. 



NON-PROTEIN NITROGEN 183 

(b) 10 cc. of Nessler's solution. 

(c) Distilled water to make volume of 100 cc. 
14. Compare in colorimeter. 

Calculation.— Multiply 20 (the height of the standard) by 15 and 
divide by the reading of the colorimeter tube containing the unknown 
to obtain the amount of urea nitrogen in terms of mg. per 100 cc. of 
blood. 

Findings.— The normal findings are from 12 to 15 mg. urea nitrogen 
per 100 cc. of blood. An increase in the quantity (nitrogen retention) 
is seen in cases of nephritis, the amount of increase varying to some 
extent with the gravity of the process. 



NON-PROTEIN NITROGEN. 

Myers's Method (Modified from those of Folin and Denis and 
Greenwald).— Principle.— The proteins are removed by precipitation 
with trichloracetic acid, and the filtrate is boiled in the presence of 
an oxidizing agent with concentrated sulphuric acid, so that the nitro- 
genous bodies are converted to ammonium sulphate. This is decom- 
posed by the action of an alkali with the formation of ammonia, 
which is liberated by aeration and aspirated into a dilute solution of 
sulphuric acid, where ammonium sulphate is formed. The amount of 
this is determined after Nesslerization by colorimetry. 

Reagents Required.— All of those required for the determination of 
urea will be needed with the exception of urease, and in addition 
potassium sulphate, concentrated sulphuric acid, 10 per cent, aqueous 
solution of copper sulphate, trichloracetic acid (2.5 per cent, dilution 
in water). 

Procedure.— In a flask are placed 20 cc. of 2.5 per cent, trichloracetic 
acid and to it are added slowly 5 cc. of oxalated blood, stirring the 
mixture with a glass rod during the process of addition. The mixture 
is allowed to stand for thirty minutes, when it is filtered. Into the 
ignition tube is placed 10 cc. of the filtrate (equivalent to 2 cc. of blood). 
To the filtrate are added 1 cc. concentrated sulphuric acid, 1 gm. 
potassium sulphate, and 2 drops 10 per cent, solution of copper sulphate. 
The mixture is then boiled over a small micro flame. Provision should 
be made for the removal of fumes as shown in the illustration (Fig. 56). 
Heating should be allowed to proceed until digestion is complete. It is 
necessary to evaporate practically all of the fluid. The small residue 
chars to a brown color. This must be heated still further until the 
solution is practically colorless or is a faint bluish green. The tube is 
allowed to cool for a moment or so, but not long enough to permit 
solidification of the solution, when about 3 cc. of distilled water are 
added. 

The solution is now to be treated with a strong alkali and aerated to 
aspirate the ammonia, as in the determination of urea. In the tube are 



184 



EXAMINATION OF THE BLOOD 



placed 5 cc. of a saturated solution of sodium carbonate and 1 cc. of 
amyl alcohol and the rubber stopper is inserted tightly. A cylindrical 
100 cc. graduate should have been charged previously with 2 cc. of 
decinormal sulphuric acid and 15 cc. of distilled water. The ammonia 
liberated from the test-tube is aspirated by the air current into the 
dilute sulphuric acid. Aeration should be allowed to proceed for 
thirty to forty-five minutes. At the end of this time Nesslerization, 
the preparation of the standard solution, the reading of the result, 
and the calculation may be carried out as described for the deter- 
mination of urea in the blood (q. v.). 



Gas 




To Vacuum 



Pump 









i 


v 




\ 


_~_~ ' 


:. ~ — _-_~_ 


_ - _ . 


— - - - 






_~_~_ _ 


.-- ZZ z — 


~-~~-~--k 


ii=$. 



-Arrangement for apparatus for determination of non-protein nitrogen of blood 
with arrangement for removing fumes. (After Myers.) 



Resume of Method.— 

1 . Place 20 cc. of 2.5 per cent, trichloracetic acid in a small flask. 

2. Add slowly 5 cc. of oxalated blood, stirring with glass rod after 

each addition. 

3. Let stand thirty minutes, then filter. 

4. Place 10 cc. of filtrate in ignition tube. 

5. Add (a) 1 cc. concentrated sulphuric acid. 

(b) 1 gm. potassium sulphate. 

(c) 2 drops of 10 per cent, copper sulphate. 

C. Boil tube over small micro flame until brown color changes to white 
or faint bluish-green. 



NON-PROTEIN NITROGEN 185 

7. Cool for a moment or two, avoiding solidification. 

8. Add 3 cc. distilled water. ■ 

9. Place tube inside cylinder A of the aerating outfit. 

10. In receiving cylinder B, place 2 cc. of jjy H2SO4 and 15 cc. of 

distilled water. 

11. Add to tube in cylinder A, 5 cc. of saturated solution of sodium 

carbonate. 

12. Stopper cylinders A and B connect up the apparatus, and start 

the air current. 

13. Allow aeration to proceed for thirty to forty-five minutes. 

14. Take cylindrical graduate C of the same size as B and mix in it 

the standard ammonium sulphate solution. 
Treat the contents of cylinders B and C as follows: 

cylinder b (contains the cylinder c (contains the 

unknown). standard). 

15. Contains substances placed in Place in cylinder 5 cc. standard 

it in step 10 plus the liber- solution, ammonium sulph- 

ated ammonia. ate (contains 1 mg. nitrogen). 

16. Add 20 cc. distilled water. 

17. Add 20 per cent, dilution of Add 20 cc. of 20 per cent, dilu- 

Nessler's solution to maxi- tion of Nessler's solution, 

mum color. 

18. Dilute to 30, 50, or 100 cc. to Bring total volume to 100 cc. 

match roughly the color of C. with distilled water. 

19. Compare contents of two cylinders in colorimeter. 

Folin and Wu's Method. — Principle.— Protein-free filtrate is treated 
with an acid digestion mixture by means of which the nitrogen bodies 
are broken down. The resulting solution is directly Nesslerized and 
compared with a standard nitrogen solution, similarly Nesslerized. 

Reagents Required. — 1. Sodium Tungstate.—See page 173. 

2. Two-thirds Normal Sulphuric Acid. 

3. Sulphuric-phosphoric Acid Digestion Mixture.— 300 cc. of phos- 
phoric acid syrup (about 85 per cent. H3PO4) are mixed with 100 cc. 
of concentrated sulphuric acid in a tall cylinder. This is covered 
to prevent the absorption of ammonia and set aside to permit sedi- 
mentation of calcium sulphate. Sedimentation is slow, but in a 
week or so the top part is clear enough to permit the removal of 50 
or 100 cc. by means of a pipette. To 100 cc. of the clear acid mixture 
is added to 10 cc. of 6 per cent, copper sulphate solution and 100 
cc. of water. 

4. Standard Nitrogen Solution.— 5 cc. containing 3 mg. of nitrogen- 
See page 180. 

5. Nessler's Solution.— Folin and Wu's formula. See page 180. 



186 EXAMINATION OF THE BLOOD 

Procedure.— Blood is obtained and oxalated as for other chemical 
procedures (see page 167). Protein is precipitated by sodium tung- 
state and sulphuric acid as described previously (see page 173). Five 
cc. of the filtrate (corresponding to 0.5 cc. of blood) are transferred 
to a dry clean'Pyrex ignition tube having a capacity of about 75 cc. 
(200 X 25 mm.). The tube should be graduated at 35 and 50 cc. by 
marks on two sides. Folin emphasizes the importance of having the 
tube thoroughly dry to prevent bumping. One cc. of the sulphuric- 
phosphoric acid mixture is added, a dry quartz pebble placed in the 
tube, and the mixture boiled over a microburner until the character- 
istic dense acid fumes fill the tube. These are usually seen in from 
three to seven minutes. When definitely present, the size of the flame 
is reduced so that the contents are just boiling and the mouth of the 
flask closed with a small watch glass or tiny Erlenmyer flask, after 
which the boiling is continued for two additional minutes. When 
solution is almost colorless, it should be boiled until practically all 
trace of color has disappeared. The contents are cooled for seventy 
to ninety seconds and 15 to 25 cc. of water are added. After cooling 
further to about room temperature, water is added to exactly the 35 
cc. mark. 

The standard should be prepared now, placing 5 cc. of the nitrogen 
standard in 100 cc. volumetric flask, adding in turn 2 cc. of the sul- 
phuric-phosphoric acid mixture, 50 cc. of water, and 30 cc. of Nessler's 
solution (Folin and Wu's formula). The flask is filled with water 
to the 100 cc. mark and the contents are mixed. 

15 cc. of Nessler's solution are added to the unknown in the tube. 
Should turbidity develop, a portion is centrifuged and the supernatant 
clear fluid compared in the colorimeter with the unknown. 

Resume of Method.— (Steps 1 to 3 inclusive are the same as those 
used for securing j)rotein-free filtrate for blood-sugar determinations.) 
Amount of blood treated depends upon number of tests to be done. 

1. Place 1 volume of well-mixed, oxalated blood in an Erlenmyer flask. 

2. Add (a) 7 volumes of distilled water. 

(6) 1 volume of 10 per cent, sodium tungstate. 
(c) 1 volume of two-thirds normal H0SO4. 
Rotate flask constantly while making additions. 

3. Filter when coagulation is complete. 

4. Transfer 5 cc. of filtrate to dry 75 cc. graduated ignition tube. 

5. Add a dry quartz pebble; boil vigorously over microburner until 

dense acid fumes fill test-tube. 

7. Reduce flame so as to keep at boiling point only; close test-tube 

mouth with watch-glass. 

8. Continue heating two minutes. (Solution must be nearly color- 

less.) 

9. Cool seventy to ninety seconds; add 15 to 25 cc. of water. 

10. Cool to about room temperature and add water to 35 cc. mark. 
Prepare standard and unknown as follows: 



BLOOD CREATININE 187 

Tube with Unknown. Flask with Standard. 

11. 5 cc. of standard nitrogen solu- 

tion (= Q.3 mg. N.). 

12. Add 2 cc. of sulphuric-phos- 

phoric acid mixture. 

13. Add 50 cc. of water. 

14. Add 15 cc. of Nessler's solution. Add 30 cc. of Nessler's solu- 

tion. 

15. If turbid, centrifuge a portion Bring to 100 cc. mark with 

and decant clear fluid for distilled water and mix. 

comparison. 

16. Compare in colorimeter. 

Calculation.— The reading of the standard is divided by the reading 
of the unknown and multiplied by 30 to give the grams of non-protein 
nitrogen per 100 cc. of blood. 

Findings.— Normally 25 to 30 mg. of non-protein nitrogen are 
present in 100 cc. of blood. The amount may be increased in the same 
conditions which lead to retention of urea in the blood (q. v.). In 
fact, many workers prefer not to estimate the non-protein nitrogen 
but rather to estimate the urea in the blood, since the latter comprises 
an almost constant proportion of the former and the procedure for its 
determination is much simpler. 

BLOOD CREATININE. 

Myers and Lough's Method (Modified from Folin). — Principle.— The 
proteins are precipitated from the laked blood by saturation with 
picric acid. They are removed by centrifugalization and filtering, 
and to the filtrate is added a dilute alkali. This results in the forma- 
tion of sodium picrate, which is reduced by the creatinine. The 
amount of creatinine is determined by the amount of the color change, 
compared with the effects of the same amount of alkali on picric 
acid solutions of creatinine of known strength. 

Reagents Required.— 1. Standard solutions of creatinine three in 
number, made up to contain respectively 0.3 mg., 0.5 mg. and 1.0 mg. 
to 100 cc. of a saturated solution of picric acid. 

2. Ten per cent, aqueous solution of sodium hydrate. 

3. Picric acid, pure, crystals. 

For method of testing purity of picric acid and for purifying it, see 
page 442. For method of preparing creatinine standards, see page 
444. 

Procedure.— Blood is obtained and the proteins are precipitated by 
saturation by the addition of picric acid crystals, as directed in the 
method for the determination of sugar (see page 169). The proteins 
are separated by centrifugalization and filtration in the same way. If 
both sugar and creatinine determinations are to be made, portions of 
the same filtrate may be used for the two tests. Into a 10 cc. graduated 



188 



EXAMINATION OF THE BLOOD 



cylinder (Fig. 54, i) are placed 10 cc. of the filtrate and into each of the 
other three cylinders are placed 10 cc. of different standard solutions, 
containing respectively, 0.3 mg., 0.5 mg., and 1.0 mg of creatinine to 
100 cc. of saturated picric acid solution. To each cylinder is then 
added 0.5 cc. of a 10 per cent, aqueous solution of sodium hydrate. 
The contents of the cylinders are mixed by placing the thumb over 
the ends and inverting gently, when they are allowed to stand for 
exactly eight minutes, at the end of which time the reading should 
be made promptly. That standard should be selected whose shade most 
closely approximates that of the unknown. The standard is then 
placed in the left hand cup of the colorimeter and the unknown in the 
right hand cup. With the cup containing the standard set at 10 mm., 
the reading is taken of the unknown. 
Resume of Method.— 

1. To 5 cc. of blood in heavy-walled 50 cc. centrifuge tubes add 20 cc. 

of distilled water. 

2. Add dry picric acid to saturation stirring thoroughly with glass rod. 

3. Let stand five minutes, then centrifugalize. 

4. Filter. 

5. Place 10 cc. filtrate in a 10 cc. graduate cylinder. Use three other 

graduated cylinders of same size for three standard solutions. 



CYLINDER A 

(Contains the 
unknown) . 



Place 10 cc. of 
filtrate. 



CYLINDER B. 

(0.3 mg. crea- 
tinine per 
100 cc). 

Place 10 cc. of 
proper stan- 



CYLINDER C. 

(0.5 mg. crea- 
tinine per 
100 cc). 

Place 10 cc. of 
proper stan- 



CYLINDER D. 

(1.0 mg. crea- 
tinine per 
100 cc). 

Place 10 cc. of 
proper stan- 



dard solution. dard solution. dard solution. 

". Add 0.5 cc. 10 Add 0.5 cc. 10 Add 0.5 cc. 10 Add 0.5 cc. 10 
per cent Na- per cent. Na- per cent. Na- per cent. Na- 
OH solution. OH solution. OH solution. OH solution. 

>. Put thumb over end of each cylinder in turn, invert to mix, and 
let all stand exactly eight minutes. Then select standard whose 
color most nearly matches that of unknown. Compare this 
with the unknown (cylinder A) in the colorimeter. 

Calculation.— The result may be determined by the formula 



(SA) 



when S represents the reading of the standard; R, the reading of the 
unknown; A, mg. creatinine in 100 cc. of the standard; and X, mg. 
creatinine in 100 cc. of blood. 

Folin and Wu's Method.— Principle.— A protein-free filtrate is 
obtained after coagulation with tungstic acid. This is treated with 



BLOOD CREATININE 189 

a solution of picric acid in the presence of an alkali and the color change 
produced is compared with that caused by a standard solution of crea- 
tinine on a similar alkaline picrate solution. 

Reagents Required.— 1. Sodium Tungstate (10 per cent, solution). 

2. Two-thirds Normal Sulphuric Acid. 

3. Saturated Solution of Picric Acid.— See page 442. 

4. Sodium Hydrate (10 per cent, solution). 

5*. Standard Creatinine Solution.— See "Preparation of Creatinine 
Zinc Chloride," page 444, for the preparation of the basic solution. 
This is made by dissolving 1.6106 gm. creatinine zinc chloride in 
sufficient decinormal hydrochloric acid to make 1 liter; 1 cc. of this 
solution contains 1 mg. of creatinine; 6 cc. are placed in a liter flask 
with 10 cc. of normal hydrochloric acid. Water is added to bring 
the volume to 1000 cc. The contents of the flask are mixed and 
transferred to a bottle for storage, adding 4 or 5 drops of xylene or 
toluene. 5 cc. of this solution contains 0.03 mg. of creatinine and when 
diluted with 15 cc. of water as directed below, furnish a standard 
suitable for most blood specimens. When there is marked retention, 
10 cc. of standard may be diluted with 10 cc. of water, the standard 
now containing 0.06 mg.; or 15 cc. with 5 cc. of water, the standard 
containing 0.09 mg. 

Procedure.— Blood is obtained and oxalated as for other chemical 
procedures (see page 167). Protein is precipitated by sodium tung- 
state and sulphuric acid as described previously (see page 173); 25 
cc. of a saturated solution of picric acid (see page 442) are placed in 
a small clean flask and mixed with 5 cc. of 10 per cent, sodium hydrate. 
Then two small flasks are taken. In one are placed 10 cc. of the pro- 
tein-free blood filtrate (corresponding to 1 cc. of blood), while in the 
other are placed 5 cc. of the standard creatinine solution, containing 
0.03 mg. creatinine and 15 cc. of water. Next 5 cc. of freshly prepared 
alkaline picrate are added to the creatinine solution. After standing 
eight to ten minutes, the contents of the two flasks are compared in 
the colorimeter. 

Resume of Method.— (Steps one to three inclusive are the same as 
those used for securing protein-free filtrate for blood-sugar determina- 
tions, see page 173. (Amount of blood treated depends upon number 
of tests to be done.) 

1. Place 1 volume of oxalated blood in an Erlenmyer flask. 

2. Add (a) 7 volumes of distilled water. 

(b) 1 volume of 10 per cent, sodium tungstate. 

(c) 8 volumes of two-thirds normal sulphuric acid. 
Rotate flask constantly while making additions. 

3. Filter when coagulation is complete. 

4. Employ a portion of filtrate as shown below (Step 6). 

5. Make fresh alkaline picrate solution by mixing 25 cc. of saturated 

solution picric acid with 5 cc. sodium hydrate, 10 per cent. 
Use in Step 8 as shown below. 



190 EXAMINATION OF THE BLOOD 

Flask A Flask B 

(Contains Unknown). (Contains Standard). 

6. Place in flask 10 cc. of blood Place in flask 5 cc. of standard 

blood filtrate from Step 3. creatinine solution (0.3 mg.). 

7. Add 15 cc. of water. 

8. Add 5 cc. of alkaline picrate Add 10 cc. alkaline picrate solu- 

solution from Step 5. tion from Step 5. 

9. Let stand eight to ten minutes. 
10. Compare in colorimeter. 

Calculation.— Multiply the reading of the standard by 1.5 and divide 
by the reading of the unknown to obtain the number of milligrams of 
creatinine per 100 cc. of blood. If the double strength were employed, 
this result should be multiplied by two. 

Findings.— The normal findings are from 1 to 2 mg. creatinine in 
100 cc. of blood. Myers and Lough concluded that the creatinine 
very seldom rose above 2.5 mg. per 100 cc. of blood unless there were 
renal involvement. Creatinine values of from 3.0 to 5 mg. should be 
regarded as decidedly unfavorable, and figures over 5.0 mg. probably 
indicate an early fatal termination. 



CREATININE PLUS CREATININE. 

Folin and Wu's Method. — Reagents Required.— The same as for the 
determination of creatinine by the Folin and YYu method and, in 
addition, normal hydrochloric acid. 

Procedure. — Five cc. of the protein-free blood filtrate obtained 
after coagulation with tungstic acid (see page 173) are placed in a 
test-tube graduated at 25 cc. 1 cc. of normal hydrochloric acid 
is added and the tube heated in an autoclave, either at 130° C. for 
twenty minutes, or at 155° C.for ten minutes after covering the mouth 
with tin-foil. After cooling 5 cc. of alkaline picrate solution are 
added. At the same time the standard should be prepared by placing 
20 cc. of creatinine standard solution in a 50 cc. volumetric flask, 
mixing with it 2 cc. of normal hydrochloric acid and 10 cc. of alkaline 
picrate solution. Both standard and unknown are allowed to stand 
for eight to ten minutes, when water is added to bring the volume of 
the unknown to 25 cc. and that of the standard to 50 cc. Comparison 
is made immediately in the colorimeter. 

Calculation.— The reading of the standard, multiplied by 6 and 
divided by the reading of the unknown, gives the amount of total 
creatinine in mg. per 100 cc. of blood. 

Normal Findings .—The authors state that the normal value for 
"total creatinine" as determined by this method is about 6 mg. per 
100 cc. of blood. 



URIC ACID 191 



URIC ACID. 



Folin and Wu's Method. — Principle.— The proteins are removed 
from the blood by filtration after the precipitation with tungstic acid 
and the purins in the filtrate are then precipitated with silver lactate. 
The uric acid is set free from the precipitate by dissolving it in acidu- 
lated 10 per cent, sodium chloride solution. On the addition of phos- 
photungstic acid solution (Folin-Denis uric acid reagent), uric acid 
gives a delicate color reaction, which is used as a quantitative test. 
For the purpose of comparison a standard solution containing a known 
amount of uric acid is similarly treated. 

Reagents Required.— 1. Folin-Denis Uric Acid Reagent (Benedict's 
modification) : 

Sodium tungstate 100 gm. 

Hydrochloric acid (concentrated) 20 cc. 

Phosphoric acid (85 per cent.) 30 " 

Distilled water q. s 1000 " 

The sodium tungstate, hydrochloric acid, and phosphoric acid are 
boiled with 750 cc. of water for two hours, preferably under a reflux 
condenser. After cooling, the volume is made up to a liter with dis- 
tilled water. 

2. Folin s Silver Lactate Solution.— This is a 5 per cent, solution of 
silver lactate in 5 per cent, solution lactic acid. 

3. Potassium Cyanide.— K 5 per cent, solution in water. Folin 
states that sodium cyanide may be used instead. 

4. Sodium Tungstate (10 per cent, solution).— See page 173. 

5. Sulphuric Acid Solution, Two-thirds Normal. 

6. Standard Uric Acid Sulphite Solution.— Make 1 to 3 liters of 
20 per cent, solution of sodium sulphite, allow to stand overnight, 
and filter. Dissolve 1 gm. uric acid in about 125 cc. of 0.4 per cent, 
lithium carbonate solution and dilute to a volume of 500 cc. Transfer 
portions of 50 cc. each to a series of volumetric flasks of 1 liter capacity. 
This amount contains 100 mg. of uric acid. To each flask add 200 
to 300 cc. of water, then 500 cc. of filtered sodium sulphite solution, 
and finally make up to volume with water. Mix well. Fill with the 
solution a series of 200 cc. bottles, stoppering tightly with rubber 
stoppers. Folin has found that the solution in a bottle which is 
opened daily will keep at least three to four months. 

7. Sodium Sulphite (10 per cent, solution) .—The surplus 20 per 
cent, sodium sulphite solution left after preparing the above uric 
acid standard is diluted with an equal amount of water to give a 
concentration of 10 per cent, and is transferred to a series of small, 
tightly stoppered bottles. This is added to the unknown to offset 
the sulphite content of the standard. 

8. Sodium Carbonate (20 per cent, solution). 

9. Sodium Chloride Solution (10 per cent, solution).— This is pre- 
pared by dissolving the salt in decinormal hydrochloric acid. 



192 EXAMINATIOX OF THE BLOOD 

Procedure.— The proteins in a specimen of blood are precipitated 
with tungstic acid according to the method described elsewhere (see 
page 173). 

Twenty cc. of the filtrate are now transferred to a large 50 cc. centri- 
fuge tube. If a centrifuge (Fig. 57) of this capacity be not available, 
two small 15 cc. tubes may be used, placing 10 cc. of the filtrate in each 
tube; or the amount may be run down in two lots of 10 cc. each. 4 cc. 
of 5 per cent, silver lactate solution in 5 per cent, lactic acid are added 
to 20 cc. of filtrate, or 2 cc. to each 10 cc. amount. The mixture is 
stirred and centrifugal i zed. A drop of silver lactate solution is added 
to the supernatant fluid, which should be practically clear and should 
show no turbidity on the addition of the last drop of the silver solution. 




Fig. 57. — Type of centrifuge suitable for serological work. 

The supernatant fluid is removed by decantation as thoroughly as 
possible and discarded. To the precipitate from 20 cc. are added 
2 cc. of 10 per cent, solution of sodium chloride in y^ HC1. If the 
two portions of 10 cc. be used, then 1 cc. should be added to each por- 
tion. This is stirred well with a glass rod, about 5 or 6 cc. of water 
are added, again stirred, and centrifugalized. The supernatant fluid, 
containing the dissolved uric acid, is poured into an accurately gradu- 
ated 25 cc. volumetric flask. If the precipitate were run down in 
two lots of 10 cc. each, the fluids should be mixed. Now should be 
added in order 1 cc. of 10 per cent, sodium sulphite solution, 0.5 cc. 
of 5 per cent, sodium cyanide solution, and 3 cc. of 20 per cent, sodium 
carbonate solution. 



URIC ACID 



193 



Simultaneously two standard uric acid solutions should be pre- 
pared as follows: Transfer to two 50 cc. volumetric flasks 1 cc. and 
2 cc. respectively of the standard uric acid sulphite solution. Add 
1 cc. of 10 per cent, sodium sulphite to the first flask. Then add to 
each flask 4 cc. of acidified salt solution, 1 cc. of 5 per cent, sodium 
cyanide solution, and 6 cc. of sodium carbonate solution, diluting with 
water to about 45 cc. 

When the two standards and the unknown have been prepared in 
this way, they are ready for the addition of Folin and Denis's uric 
acid reagent, of which 0.5 cc. is added to the unknown and 1 cc. to 
the standards. The contents are mixed and after standing for ten 
minutes, all flasks are filled to the marks with water and again mixed, 
when the unknown is compared in the colorimeter with the standard 
which matches more nearly in color. 

Resume of Method.— (Steps 1 to 3 inclusive are the same as those 
used for securing protein-free filtrate for blood-sugar determinations 
see page 173.) 

1. Place 1 volume of well-mixed, oxalated blood in an Erlenmyer 

flask. 

2. Add (a) 7 volumes of distilled water. 

(6) 1 volume of 10 per cent, sodium tungstate. 
(c) 1 volume of two-thirds normal H 2 S0 4 . 
Rotate flask constantly while making additions. 

3. Filter when coagulation is complete. 

4. Transfer 20 cc. of filtrate to 50 cc. centrifuge tube. 

5. Add 4 cc. of silver lactate solution and stir well. 

6. Add 5 cc. of water, stir again, and centrifugalize. 

7. Add another drop of silver lactate. Supernatant fluid should be 

clear. If cloudy, centrifugalize again. 

8. Decant and discard supernatant fluid. 

9. Add 2 cc. of acidified salt solution to sediment, stir and centrifugalize. 

10. Transfer to 25 cc. volumetric flask (flask A). Treat as shown 

below. 

11. Take two 50 cc. volumetric flasks and prepare as shown below. 



Flask A 

(Unknown) . 

12. Dissolved precipi- 

tate as shown in 
Step 10. 

13. Add 1 cc. of 10 per 

cent, sodium sul- 
phite. 
14. 

15. Add 0.5 cc. of 5 per 
cent, sodium cya- 
nide. 
13 



Flask B 

(First Standard) . 

Put in 1 cc. of uric 
acid sulphite 
standard sol. 

Add 1 cc. of 10 per 
cent, sodium sul- 
phite. 

Add 4 cc. of acidified 

salt sol. 
Add 1 cc. of 5 per 
cent, sodium cya- 
nide. 



Flask C 

(Second Standard). 

Put in 2 cc. of uric 
acid sulphite 
standard sol. 



Add4 cc. of acidified 
salt sol. 

Add 1 cc. of 5 per 
cent, sodium cya- 
nide. 



194 



EXAMINATION OF THE BLOOD 



Flask A 



Flask B 



Flask C 



16 



Add 3 cc. of 20 per 
cent, sodium car- 
bonate. 



17 



US 



Add0.5cc.ofFolin- 
Denis reagent and 
mix. 
19. Bring volume to 25 
cc. with water 
and mix. 



Add 6 cc. of 20 per 
cent, sodium car- 
bonate. 

Dilute with water 
to about 45 cc. 

Add 1 cc. of Folin- 
Denis reagent and 
mix. 

Bring volume to 50 
cc. with,water 
and mix. 



Add 6 cc. of 20 per 
cent, sodium car- 
bonate. 
Dilute with water 

to about 45 cc. 
Add 1 cc. of Folin- 
Denis reagent and 
mix. 
Bring volume to 50 
cc. with water 
and mix. 

20. Let all flasks stand ten minutes and compare in colorimeter the 
contents of A with the standard solution which matches more 
closely. 
Calculation.— The concentration of the unknown may be computed 
with this formula, when the weaker standard is used: 

2.5 S 

Mg. of uric acid per 100 cc. of blood = ■ 

R 
S represents the reading of the standard and R that of the unknown. 
When the stronger standard is used, the result obtained by the above 
formula is multiplied by 2. 

Myers's Modification of Folin's Method.— Principle.— The principle 
is essentially the same as in Folin's method except that a different 
standard is employed. 

Reagents Required.— The same as for Folin's method except that 
sodium sulphite is not needed, and that Benedict's standard is 
employed. 

Benedict's Standard Uric Acid Solution. 

Nine grams of pure crystalline hydrogen di-sodium phosphate and 
1 gram of dihydrogen sodium phosphate are dissolved in 300 cc. of 
hot distilled water. This solution should be filtered and the volume 
made up to 500 cc. with distilled water. The clear, warm solution 
is poured onto 200 mg. of uric acid, suspended in a little water in a 
volumetric flask of one liter capacity. The flask should be stoppered 
and the mixture shaken until the uric acid is completely dissolved. 
Then 1.4 cc. of glacial acetic acid is added by accurate measurement 
and the total volume is brought to one liter with distilled water. About 
5 cc. of chloroform should be added as a preservative. This solution 
should be prepared freshly about every two months. One cc. of this 
solution contains 0.2 mg. of uric acid. 

Procedure.— -The procedure is the same as for the preceding method 
up to and including the step when the precipitate obtained with 
silver lactate is treated with acidified salt solution and centrifugalized. 
After centrifugalization, the supernatant clear fluid is decanted into 
an accurately graduated 25 cc. cylinder, and the standard solution is 
prepared. 



URIC ACID 195 

With an accurate pipette, Ostwald or Folin type, 1 cc. of the uric 
acid standard is placed in another accurately graduated 25 cc. cylinder. 
Four cc. of 10 per cent, sodium chloride made up ony^HCl are added 
with enough water to make the volume essentially the same as that of 
the unknown and a drop of 5 per cent, potassium or sodium cyanide. 
To the standard there should be added 1 cc. of the Folin-Denis uric acid 
reagent and 6 cc. of saturated solution of sodium carbonate, while to 
the unknown there should be added 0.5 cc. uric acid reagent and 3 cc. 
saturated solution of sodium carbonate. The color is allowed to 
develop for five minutes, when the standard is diluted to 25 cc. with 
distilled water. The unknown is likewise diluted with distilled water 
until its color is of similar intensity. The color of the unknown is 
compared with that of the standard by the colorimeter, in the manner 
described for blood sugar, creatinine, etc. 

Resume of Method.— (Steps one to three inclusive are the same as 
those used for securing yrotein-free filtrate for blood sugar determina- 
tions.) 

1. Place 5 cc. of well-mixed, oxalated blood in Erlenmeyer flask. 

2. Add (a) 35 cc. of distilled water. 

(6) 5 cc. of 10 per cent, sodium tungstate. 
(c) 5 cc. of two-thirds normal H 2 S0 4 . 
Rotate flask constantly while making additions. 

3. Filter when coagulation is complete. 

4. Transfer 20 cc. of filtrate to large 50 cc. centrifuge tube. 

5. Add 4 cc. of 5 per cent, silver lactate solution (made up in 5 per 

cent, lactic acid). Stir well. 

6. Add 5 or 6 cc. of water; stir again. 

7. Centrifugalize. 

8. Pour off and discard supernatant fluid. 

9. To the precipitate add 2 cc. of 10 per cent, sodium chloride (made 

upin T N o HC1). 

10. Stir with glass rod, add 5 or 6 cc. water, and stir again. 

11. Centrifugalize. 

12. Pour supernatant fluid into graduated 25 cc. cylinder (A). Treat 

this as outlined below. Sediment is discarded. 

13. Take a similar 25 cc. graduated cylinder and in it prepare standard 

as outlined below. 



CYLINDER A (UNKNOWN) . CYLINDER B (STANDARD) . 

14. Contents as directed in step 13. Put in 1 cc. of standard uric 

acid solution. 

15. Add 4 cc. of 10 per cent. NaCl 

(made up in -^o HO). 

16. Add distilled H 2 to make vol- 

ume same as in cylinder A. 



196 EXAMINATION OF THE BLOOD 

CYLINDER A ( UNKNOWN) . CYLINDER B (STANDARD). 

17. Add a drop of 5 per cent. Add a drop of 5 per cent. 

NaCN solution. NaCN solution 

18. Add 0.5 cc. of Folin-Denis uric Add 1 cc. of Folin-Denis uric 

acid reagent. acid reagent. 

19. Add 3 cc. of saturated solution Add 6 cc. of saturated solution 

Na 2 C0 3 . Na s CO,. 

20. Allow to stand five minutes. Allow to stand five minutes. 

21. Add water to approximately Add water to bring volume to 

match intensity of color in 25 cc. 

standard which has been 
diluted to 25 cc. 

22. Compare contents of two cylinders in colorimeter. 

Calculation.— The concentration of the unknown solution may be 
computed with the aid of the formula: 

4SA 
X _ 5RB 

X is the number of mg. uric acid per 100 cc. of blood, while S repre- 
sents the depth of colorimeter cup containing the standard, R the 
reading of the unknown, A the number of cc. to which the unknown was 
diluted for color comparison and B the number of cc. of blood repre- 
sented by the amount of filtrate employed. 

Findings.— The normal findings run from 2 to 3 mg. per 100 cc. of 
blood. The amount is increased in gout. The determination of uric 
acid has been advocated as a valuable guide in early nephritis, since 
an increase is seen in the blood before the other non-protein nitrogen 
elements show elevation over normal figures. Amounts as high as 
12 mg. per 100 cc. of blood in early interstitial nephritis have been 
reported. 

GENERAL CONSIDERATIONS REGARDING THE RETENTION 
OF URIC ACID, UREA AND CREATININE. 

In gout the blood shows a high uric content. This is seen also in 
cases of early nephritis, though in such instances there is usually an 
increase in the amount of urea also. Myers and Chace have studied 
the retention of the various nitrogenous factors in cases of nephritis 
and describe what they term the "staircase retention." According 
to their investigations, uric acid increase is seen in the less severe 
cases. In the next group, the blood urea is increased as well as the 
uric acid, while in the most severe type, marked by fatal termination, 
there is an increase in the creatinine as well as in the urea and uric 
acid. Myers states that the creatinine is more easily eliminated than 
uric acid or urea and that there is usually not an appreciable increase 
in the blood until the blood urea has doubled, or more than doubled, 
the normal amount. 



REGARDING THE RETENTION OF URIC ACID 



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198 EXAMINATION OF THE BLOOD 



TESTS FOR ACID INTOXICATION. 

Acidosis has been defined as a depletion of the blood and other 
tissues of the body in fixed bases (Sellards). It may result from 
the defective oxidation of abnormally formed organic acids as in 
diabetes, or from the decreased elimination of acid substances which 
are normally present, as in nephritis. The "alkali reserve" of the 
serum, by means of which acids are neutralized, is made up of the 
bicarbonates, small quantities of phosphates, and alkaline protein 
compounds. Under normal conditions these substances are present 
in quite constant quantities and reaction is maintained in a state of 
constant equilibrium. An acid product normally present is carbon 
dioxide. This is taken up by the serum partly in solution and partly 
in combination with the carbonates. When it is increased in amount, 
there is an overstimulation of the respiratory center with a resultant 
increase in pulmonary ventilation and a greater loss of carbon dioxide 
so that the reaction of the blood is kept for a time within normal limits, 
and there is no draft upon the "alkali reserve." With increasing 
amounts of acid, however, non-volatile acids combine with the car- 
bonates. These cannot be eliminated by increasing pulmonary 
ventilation and they combine with the carbonates, which are called by 
Henderson the "first line of defense" and encroach upon the alkali 
reserve. For a time urinary excretion may serve as a compensatory 
mechanism since the kidneys have the power of excreting an acid urine 
from a neutral blood, thus eliminating acid. It has been shown that 
this mechanism fails in certain forms of nephritis so that the inorganic 
phosphates of the serum are increased. In acidosis, even the normal 
amount of carbon dioxide lowers the already depleted alkali reserve. 
As a consequence the respiratory center is greatly overstimulated, 
hyperpnea results, and the carbon-dioxide tension of the alveolar air 
is greatly reduced. 

Consideration of the Methods.— Several methods have been proposed 
for the determination of the degree of acidosis. Chief among these are 
the determinations of: (1) diminished carbon-dioxide combining power 
of the blood; (2) diminished carbon-dioxide tension of the alveolar air; 
(3) increased tolerance to sodium bicarbonate; (4) change in the 
reaction to phenolphthalein, from alkaline to neutral, of the protein- 
free filtrate of the blood serum; (5) increase in the hydrogen-ion con- 
centration of the blood; (6) appearance of acetone bodies in the urine; 
(7) increase in the output of urinary ammonia. 

The appearance of acetone bodies in the urine (6) can be expected 
only in carbohydrate acidosis, and the increase in urinary ammonia 
(7) is usually not found in nephritic acidosis. It is rather generally felt 
that the determination of hydrogen-ion concentration of the blood (5) 
is not a clinical method of great value on account of the obvious diffi- 
culties with colorimetric determinations made upon blood-serum or 



TESTS FOR ACID INTOXICATION 199 

even its dialysate and because it has been shown that no decided change 
occurs except in advanced cases (Sellards) . The change in reaction in 
a protein-free filtrate has been commended by Sellards, who proposed 
this test, as being not only of diagnostic value but also of considerable 
help in regard to therapy. The determination of the C0 2 combining 
power of the blood (1) and of the carbon-dioxide tension of the alveolar 
air (2), and of the tolerance of sodium bicarbonate (3) have been of 
especial clinical value. Of the first two methods the determination of 
the carbon-dioxide combining power is much more reliable and is 
carried out without any especial difficulty. Though the determination 
of the carbon-dioxide tension of the alveolar air is a simple bedside 
procedure and is of marked clinical value, the information is not as 
reliable as that obtained by the determination of the C0 2 combining 
power of the blood . The bicarbonate tolerance test is the simplest of all . 

The methods which we have designated as first, fourth, and fifth 
have been described in the section of the chapter which follows immedi- 
ately. The technic for the second method has been given in the 
chapter on miscellaneous methods and the third method, together 
with a consideration of the acetone bodies and the output of urinary 
ammonia, will be found in the chapter on urine. 

Determination of the Carbon-dioxide Combining Power of the Blood 
Plasma (method of Van Slyke and Cullen).— Principle of Method.— 
Oxalated blood is obtained by venipuncture, the plasma is separated 
by centrifugalization and thoroughly saturated with carbon dioxide. 
The carbon dioxide combines with the fixed alkalies of the plasma to 
form bicarbonates. The plasma is placed in an especially devised 
pipette, where the carbon dioxide is liberated by the action of sulphuric 
acid in the presence of a vacuum. The watery solution is then removed 
from the pipette and the liberated C0 2 gas is measured over mercury 
in the calibrated portion. It is evident that the amount of carbon 
dioxide which will be taken up by the blood and subsequently liberated 
depends upon the amount of fixed alkali in the plasma, and therefore 
that the C0 2 combining power will serve as an indication of the alkali 
reserve. 

Apparatus and Reagents Required. — 1. Van Slyke's apparatus. This 
is manufactured by the Emil Greiner Company, 55 Fulton Street, 
New York City. It should be mounted in a suitable support. The 
manufacturer ordinarily supplies a frame of wood which is held upon an 
iron tripod. 

2. Mercury sufficient to fill the apparatus and allow a moderate 
surplus. About 2 kg. will be required. 

3. Separatory funnel, holding about 100 times the volume of the 
plasma to be saturated. It is convenient to have a funnel of 250 cc. 
capacity. 

4. Ostwald pipettes, one of 1 cc. and one of 0.5 cc. capacity. 

5. Apparatus for removing moisture from the exhaled air. A glass 



200 EXAMINATION OF THE BLOOD 

bottle should be filled with glass beads and fitted with inlet and outlet 
tubes as shown in Fig. 58. 

6. Apparatus for withdrawing blood by venipuncture. 

7. Dropping bottles containing the following: 

(a) Phenolsulphonephthalein solution, 1 gm. dissolved in 100 cc. of 
95 per cent, alcohol. 
(6) Caprylic alcohol. 

(c) Sulphuric acid, 5 per cent, solution. 

(d) Ammonia, 1 per cent, concentration. The ammonia should be 
freed from carbonate by adding a small amount of barium hydrate. 
The precipitated barium carbonate can be filtered off, when the excess 
of barium is precipitated with a little ammonium sulphate. 

8. Medicine droppers, beaker containing distilled water, etc. 
Obtaining the Blood.— Blood may be obtained by venipuncture from 

an arm vein. It should be drawn directly into a dry and chemically 
clean centrifuge tube into which should have been placed enough 
powdered potassium oxalate to make about 0.5 per cent, of the weight 



K 



Fig. 58. — Apparatus for saturating plasma with C0 2 . A, separatory funnel; B, bottle 
containing glass beads; C, mouth-piece through which air is expired. 

of the amount of blood to be obtained. For withdrawing the blood, 
either the MacRae needle or the one described by the writer may be 
used. The technic of venipuncture has been described fully on page 
126. It is desirable to avoid stasis of the blood so that the use of a 
tourniquet should be dispensed with when possible. If indispensable, 
it should be released as soon as the vein has been entered and a few 
seconds should be allowed to elapse so that the stagnant blood may 
pass before the specimen is withdrawn. After the specimen has been 
obtained, the tube is stoppered with a clean cork and turned on its 
side and back to the vertical position once or twice to ensure thorough 
mixing with the oxalate. The blood is then subjected to no further 
agitation and is centrifugalized in the same tube as soon after with- 
drawal as possible to separate the serum. 

The patient should have been instructed to avoid vigorous muscular 
exercise for at least an hour before the withdrawal of the specimen. 

After centrifugalization, the supernatant clear serum is pipetted 
off with a capillary pipette. When the determination is to be made 
immediately, the serum may be placed directly into the separatory 



TESTS FOR ACID INTOXICATION 201 

funnel when the plasma has been separated at once, immediately put 
into paraffined tubes and kept on ice, it has been shown that it will 
retain its carbon-dioxide capacity for over a week without alteration. 

Saturation of Plasma with Carbon Dioxide.— The plasma is placed in 
a separatory funnel (Fig. 58, A). This is placed on its side and the 
contained air displaced by exhaling into it the alveolar air from the 
operator's lungs. This air is passed through a bottle containing glass 
beads to free it from moisture (Fig. 58, B) . The operator should not 
inspire more deeply than normal and should expire as quickly and 
completely as possible. The stopper in the funnel should be inserted 
just before the inspiration is finished, so that air will not be drawn 
back into the funnel. The stopcock is turned to seal the chamber 
at both ends, and the funnel is turned end over end for two minutes in 
order to distribute the plasma in as thin a layer over the inner surface 
of the funnel as possible. Two minutes are required for saturation. A 
number of sera may be saturated at one time, using different funnels 
for each serum, and standing them upright in a rack until ready for 
the actual determination. 

When plenty of serum is available, 3 cc. are saturated in the funnel. 
This will yield enough for two determinations. When the supply is 
limited, 0.5 cc. may be used in a determination, saturating the serum 
available in a 50 cc. funnel. In this case, the total volume of water, 
serum, and acid used in the determination should not exceed 1.25 cc, 
instead of 2.5 cc. as prescribed when a 1 cc. quantity of serum is utilized. 
When a 0.5 cc. quantity is employed, the volume of gas should be 
multiplied by two before making the corrections. 

After saturation is completed, the funnel is stood in an upright 
position until the serum drains to the bottom of the chamber, from 
which it may be taken up with the Ostwald pipette of the proper 
capacity (Fig. 54, d). In delivering the serum in cup b of the appa- 
ratus, it should be allowed to run in under the level of the dilute 
ammonia in the cup. 

Preparation of the Apparatus.— The apparatus (Fig. 59) is com- 
pletely filled with mercury, including the capillaries a and b above the 
cock e. The mercury is run into the instrument through the leveling 
bulb. To fill capillary b, the leveling bulb should be in position 1, the 
cock e should be opened to the position X', and the cock / should be 
placed first in the position Y and then in the position Y' to allow the 
mercury to rise first through c and then through d to fill A and partly 
fill b and so that all the air will be forced out. When b has been 
partly filled, cock e is turned to position X so that some of the mercury 
may run out through a into suitable vessel, filling A. 

The apparatus should be tested for tightness and freedom from gases. 
The cock e should be closed and the leveling bulb lowered to position 3, 
stopcock / being open, connecting bulb A and smaller bulb d. 'A tor- 
ricellian vacuum is obtained by allowing the mercury to fall to about 
the middle of bulb d. The leveling bulb is raised quickly to position 1. 



202 EXAMINATION OF THE BLOOD 

A sharp click will be heard as the mercury strikes the upper stopcock 
provided that there is not a cushion of air or water between the stop- 



Position 1- 



Position Z. 



Posit ion X 





Position X 




inr 



Position Y. Position Y ' 
d. 



Position 3. 
should be 
80cm.AetoYT^A 

position 2. 

FlG 59 —Van Slyke's apparatus for determination of CO, combining power of pU 
(Figure modified from Van Slyke's.) 



TESTS FOR ACID INTOXICATION 203 

cock and the upper surface of the column of mercury. If there be any 
fluid or air, it must be evacuated by turning cock e to position X and 
draining it out through outlet a, when the outlet should be closed and 
the apparatus again tested. Van Slyke has called attention to the fact 
that before the apparatus has been used it holds measurable amounts 
of gas which come from the rubber tubing and even from the glass 
itself. These gases are liberated by the vacuum. 

The stopcocks should be lubricated by a suitable stopcock grease 
(which may be obtained from the manufacturers) and held in place 
by stout elastic bands or elastic cords made of fine wire spirals. 

After using the apparatus, it is not necessary to wash it out before 
making the next determination. The cup b should be washed out with 
ammonia and the reaction should be tested with phenolphthalein to 
make certain that all the acid has been neutralized. 

When not in use, the mercury should be drained out and the appa- 
ratus should be filled with water after thoroughly greasing the cocks. 
The mercury may be cleaned occasionally by forcing it through clean 
chamois. To do this a piece of chamois about 12 inches square is held 
over a large beaker and the mercury is poured into the chamois, which 
is gathered up like a bag, with the mercury inside. The neck of the 
sack is twisted tight, the bag is squeezed, and the mercury is forced 
through the chamois into a large beaker. 

The Determination.— -The mercury is removed from cup b which is 
washed out with carbonate-free ammonia by running some in and by 
removing most of it with a medicine dropper. A trace is left and a few 
drops of water with a drop of phenolphthalein solution are added. 
If sufficient ammonia be present, the solution will be a distinct red. 
One cc. of the serum which has been saturated with carbon dioxide 
is run into the cup from an Ostwald pipette, whose tip should be kept 
beneath the level of the diluted ammonia water so that there will be 
no loss of the carbon dioxide into the air. The last of the serum should 
be ejected from the pipette by keeping the upper end tightly closed 
with the finger tip while the bulb of the pipette is grasped firmly with 
the palm of the other hand. The heat is sufficient to drive out the last 
of the serum. The leveling bulb is lowered to position 2, the cock e 
is opened to position X', so that the cup b and the 50 cc. bulb are in 
communication, and the serum is drawn into the calibrated upper 
portion of the 50 cc. chamber leaving just enough to fill the capillary 
portion of b. The cock is closed and in the cup is placed about 0.5 cc. 
of water, which is drawn into the 50 cc. chamber just as the serum was. 
This is repeated. Next a drop of caprylic alcohol is drawn in to prevent 
foaming, and finally 0.5 cc. of 5 per cent, sulphuric acid. It is not 
necessary that exactly 1 cc. of wash water and 0.5 cc. be taken, but the 
total volume of fluid drawn into the 50 cc. must not exceed 2.5 cc. as 
measured on the calibrated stem. Not a single bubble of air should 
be permitted to gain access with any of the fluids which are admitted. 
After the acid has been run in, a few drops of mercury are placed in 



204 



EXAMINATION OF THE BLOOD 



cup b to seal the capillary and the acid in the cup is washed out with 

When the solutions are all in the pipette and the stopcock is closed 
and sealed off with mercury, the leveling bulb is lowered and hung in 
position 3. The cock f is opened (position Y) so that the mercury in 
the 50 cc. portion can run into the bulb d. The mercury is allowed to 
run out until the upper surface of the mercury has reached the 50 cc. 
mark of the chamber A, when the cock is closed. The 50 cc. chamber 
is now sealed above and below. The apparatus is now detached from 



Fig 




reading of Van Slyke's apparatus. (After Van Slyke and Cullen.) 



the stand and the pipette is turned upside down and back for at least 
fifteen times to ensure obtaining an equilibrium between the 2.5 cc. 
of water solution and the 47.5 cc. of free space in the apparatus. The 
apparatus is then replaced in its support with the leveling bulb still in 
position 3. The lower stop cock is turned to position Y so that the 
watery solution may be drained into chamber d as completely as pos- 
sible, not allowing any gas to follow it. Of course, it will be impossible 
to drain the last few drops which will float as a layer on the upper 
surface of the mercury, but the error caused by the absorption 
of carbon dioxide by the water will be so small that it may be neglected 



TESTS FOR ACID INTOXICATION 205 

provided the reading be made at once. The cock is then closed, 
the leveling bulb is taken in the left hand, while with the right hand 
the cock/ is opened to position Y', to connect c with the 50 cc. chamber, 
so that the mercury will fill up the chamber, a few cc. of water floating 
on top of the column. Then the mercury bulb is held so that the upper 
surface will be on the same level as the mercury meniscus in the 
calibrated portion of the pipette (Fig. 60) and the reading is taken on 
the scale. This concludes the analysis. The corrections should be 
made for temperature and barometric pressure. 

When the determination has been completed, the lower cock is 
opened (position Y') so that the 50 cc. chamber and side tube c are in 
communication. The leveling bulb is lowered and most of the mercury 
is drawn out through c. The cock is then turned (position Y) so that 
the watery solution may be re-admitted from d to A, raising the level- 
ing bulb to position 1 . Cock e is opened (position X) forcing out the 
water solution and a little mercury through capillary A. 

Calculation.— This calculation may be carried out with the aid of the 
two tables which have been worked out by Van Slyke and Cullen. 

T> 

The volume of gas observed is multiplied by the factor =™. This 

factor may be ascertained from Table I, when the barometric pressure 
at which the determination was made is known. For example, if the 

T) 

barometric pressure were 750, the factor =^ is seen to be 0.987. 

The volume of gas observed is multiplied by this and the resultant 
figure is located in the first column of Table II, the column headed 

"Observed volume of gas X 7™-'' The line is carried across horizont- 
ally to the column which is headed by the temperature at which the 
reading was made, and the result is read off. This is expressed in 
terms of cc. of carbon dioxide bound by 100 cc. of plasma, reduced to 
degrees, and a barometric pressure of 760 m. Figures for tempera- 
tures between those which are given may be calculated. 



For convenience in the calculation the values for the ratio — ~™ — 
over the range usually encountered are given below. 



Barometer. 760 

732 0.961 

734 0.966 

736 0.967 

738 0.971 

740 0.974 

742 0.976 

744 0.979 

746 0.981 

748 0.984 

750 0.987 

752 0.989, 

754 0.992 





Barometer. 


Barometer. 


760 


756 


0.995 


758 


0.997 


760 


1.000 


762 


1.003 


764 


1.006 


766 


1.008 


768 


1.011 


770 


1.013 


772 


1.016 


774 


1.018 


776 


1.021 


778 


1.024 



206 



EXAM1XATIOX OF THE BLOOD 



TABLE II. 



-TABLE FOR CALCULATION OF CARBON DIOXIDE 
COMBINING POWER OF PLASMA. 



Observed 

vol. gas 
B 


Cc. of C0 2 reduced to 0°, 760 

mm., bound as bicarbonate by 

100 cc. of plasma. 


Observed 

vol. gas 
v B 


Cc. of C0 2 reduced to 0°, 760 

mm., bound as bicarbonate by 

100 cc. of plasma. 


K 760 


15° 


20° 25° 


30° 


X 760 


15° 


20° 


25° 


30° 


0.20 


9.1 


9.9 10.7 


11.8 


0.60 


47.7 


48.1 


48.5 


48.6 


1 


10.1 


10.9 


11.7 


12.6 


1 


48.7 


49.0 


49.4 


49.5 


2 


11.0 


11.8 


12.6 


13.5 


2 


49.7 


50.0 


50.4 


50.4 


3 


12.0 


12.8 


13.6 


14.3 


3 


50.7 


51.0 


51.3 


51.4 


4 


13.0 


13.7 14.5 


15.2 


4 


51.6 


51.9 


52.2 


52.3 


5 


13.9 


14.7 


15.5 


16.1 


5 


52.6 


52.8 


53.2 


53.2 


6 


14.9 


15.7 


16.4 


17.0 


6 


53.6 


53.8 


54.1 


54.1 


7 


15.9 


16.6 


17.4 


18.0 


7 


54.5 


54.8 


55.1 


55.1 


8 


16.8 


17.6 


18.3 


18.9 


8 


55.5 


55.7 


56.0 


56.0 


9 


17.8 


18.5 19.2 


19.8 


9 


56.5 


56.7 


57.0 


56.9 


0.30 


18.8 


19.5 20.2- 


20.8 


0.70 


57.4 


57.6 


57.9 


57.9 


1 


19.7 


20.4 


21.1 


21.7 


1 


58.4 


58.6 


58.9 


58.8 


2 


20.7 


21.4 


22.1 


22.6 


2 


59.4 


59.5 


59.8 


59.7 


3 


21.7 


22.3 


23.0 


23.5 


3 


60.3 


60.5 


60.7 


60.6 


4 


22.6 


23.3 


24.0 


24.5 


4 


61.3 


61.4 


61.7 


61.6 


5 


23.6 


24.2 


24.9 


25.4 


5 


62.3 


62.4 


62.6 


62.5 


6 


24.6 


25.2 


25.8 


26.3 


6 


63.2 


63.3 


63.6 


63.4 


7 


25.5 


26.2 


26.8 


27.3 


7 


64.2 


64.3 


64.5 


64.3 


8 


26.5 


27.1 


27.7 


28.2 


8 


65.2 


65.3 


65.5 


65.3 


9 


27.5 


28.1 


28.7 


29.1 


- 9 


66.1 


66.2 


66.4 


66.2 


0.40 


28.4 


29.0 


29.6 


30.0 


0.80 


67.1 


67.2 


67.3 


67.1 


1 


29.4 


30.0 


30.5 


31.0 


1 


68.1 


68.1 


68.3 


68.0 


2 


30.3 


30.9 


31.5 


31.9 


2 


69.0 


69.1 


69.2 


69.0 


3 


31.3 


31.9 


32.4 


32.8 


3 


70.0 


70.0 


70.2 


69.9 


4 


32.3 


32.8 


33.4 


33.8 


4 


71.0 


71.0 


71.1 


70.8 


5 


33.2 


33.8 


34.3 


34.7 


5 


71.9 


72.0 


72.1 


71.8 


6 


34.2 


34.7 


35.3 


35.6 


6 


72.9 


72.9 


73.0 


72.7 


7 


35.2 


35.7 


36.2 


36.5 


7 


73.9 


73.9 


74.0 


73.6 


8 


36.1 


36.6 


37.2 i 37.4 


8 


74.8 


74.8 


74.9 


74.5 


9 


37.1 


37.6 


38.1 38.4 


9 


75.8 


75.8 


75.8 


75.4 


0.50 


38.1 


38.5 


39.0 39.3 


0.90 


76.8 


76.7 


76.8 


76.4 


1 


39.1 


39.5 


40.0 


40.3 


1 


77.8 


77.7 


77.7 


77.3 


2 


40.0 


40.4 


40.9 


41.2 


2 


78.7 


78.6 


78.7 


78.2 


3 


41.0 


41.4 


41.9 


42.1 


3 


79.7 


79.6 


79.6 


79.2 


4 


42.0 


42.4 


42.8 


43.0 


4 


80.7 


80.5 


80.6 


80. 1 


5 


42.9 


43.3 


43.8 


43.9 


5 


81.6 


81.5 


81.5 


81.0 


6 


43.9 


44.3 


44.7 


44.9 


6 


82.6 


82.5 


82.4 


82.0 


7 


44.9 


45.3 


45.7 


45.8 


7 


83.6 


83.4 


83.4 


82.9 


8 


45.8 


46.2 


46.6 


46.7 


8 


84.5 


84.4 


84.3 


83.8 


9 


46.8 


47.1 


47.5 


47.6 


9 


85.5 


85.3 


85.2 


84.8 


0.60 


47.7 


48.1 


48.5 


48.6 


1.00 


86.5 


86.2 


86.2 


85.7 



Example.— II the observed volume of gas were 0.50, the barometric 
pressure 750 mm., and the temperature 20 degrees, the figure .50 
would be multiplied by the factor — which in consulting Table I, 
is found to be 0.987, as explained above. 0.50 times 0.987 is equal to 



TESTS FOR ACID INTOXICATION 207 

0.4935. The nearest figure to this found in Table II is 0.49. Carrying 
the line across horizontally to the column headed by the temperature 
20 degrees we find the figure 37.6. The difference between this figure 
and the one just beneath is 0.9. We take one-third of this to allow for 
the fraction expressed in the third and fourth decimal places of the 
figure 0.4935, and add it to 37.6, making our final result 37.9, which 
expresses the number of cc. of carbon dioxide bound as bicarbonate by 
100 cc. of plasma, reduced to degrees and 760 mm. pressure. 

Remarks.— On account of the added complication of making these 
computations, some workers recommend subtracting from the observed 
volume of gas for the purpose of correction the arbitrary figure 0.12. 
In the above example, for instance, 0.12 taken from the observed volume 
of gas, 0.50, would give the result 0.38, which closely approximates that 
obtained in the example. 

Normal Findings.— The bicarbonate carbon dioxide absorbed and 
yielded by the plasma amount to 0.53 to 0.75 cc. per cc. of plasma, or 
53 to 75 cc. per 100 cc. of plasma (Van Slyke and Cullen), when the 
blood has been withdrawn in the manner described. In acidosis, 
carbon dioxide capacity falls far below normal. 

Determination of the Reaction to Phenolphthalein of the Protein-free 
Blood-serum (method of Sellards). — Reagents Required.— 1. Absolute 
ethyl alcohol. The selection of alcohol which will be neutral is of 
great importance. Sellards states that it is difficult to obtain absolute 
alcohol of neutral reaction but that it is comparatively easy to obtain 
95 per cent, alcohol free from any alkali, and that preliminary tests 
do not show that this amount of water is disadvantageous. The 
alcohol should be tested. The test for alkalinity is performed by 
mixing 50 cc. of the alcohol to be tested with a drop of phenolphthalein 
and evaporating this to dryness over a water-bath. The residue should 
be colorless and should remain colorless on the addition of a drop of 
water. To test, 50 cc. are mixed with 0.1 cc. of one-hundredth normal 
sodium hydrate and evaporated to dryness with a drop of phenol- 
phthalein. The residue should turn red on drying or after a drop of 
water is added. 

Procedure.— One cc. of serum is added drop by drop to 25 cc. of tested 
alcohol in a test-tube. The proteins are precipitated. They are 
filtered off through perfectly dry filter paper in a dry funnel, taking all 
precautions to prevent the addition of any water. The precipitate is 
not washed but is evaporated to dryness over a water-bath after the 
addition of a few drops of phenolphthalein. 

Interpretation of Results.— According to Sellards, the alcoholic 
filtrates from all normal sera soon turn red and on drying, the residues, 
if kept hot, retain their color for several hours. With early acidosis 
where there was a deficit of from 20 to 30 grams of sodium bicarbonate 
(see Sellards' bicarbonate deficiency test under Urine, page 276), the 
alcoholic filtrate must become very much concentrated before the red 
color appears and the red color usually disappears soon. With more 



208 EXAMINATION OF THE BLOOD 

advanced depletion of bicarbonates, corresponding to a deficiency of 
75 to 100 gm., no red color appears at any stage of evaporation, but the 
residue becomes pink or red on adding water. With a well-marked 
acidosis corresponding to a deficit of 150 gm. of bicarbonate, the 
residue remains colorless even on adding water. 

Determination of the Alkali-reserve of the Blood Plasma (method of 
Marriott) . — Principle.— Blood serum is dialyzed against salt solution to 
remove proteins and coloring matters which would interfere with the 
exact matching of the indicators. The carbon dioxide is then removed 
from the dialysate by aeration so as to leave the unneutralized alkali 
reserve and then the hydrogen-ion concentration of the dialysate is 
determined by means of an indicator, phenolsulphonephthalein, using 
as standards for comparison phosphate solutions of varying hydrogen- 
ion concentration. Marriott states that the results compare closely 
with those obtained by the electrical method. 

Apparatus and Reagents Required.— 

1. Set of tubes containing standard phosphate mixtures. 

2. Solution of phenolsulphonephthalein in 0.8 per cent, salt solution. 

3. Collodion sacs. 

' 4. Pipette to measure 0.5 cc. 

5. Small test-tubes for aerating and dialyzing. 

6. Atomizer bulb. 

7. Glass tube or pipette drawn out to a fine capillary point. 

8. Box for color comparison, with ground glass background, on prin- 
ciple similar to Sahli hemoglobinometer. 

Preparation of Reagents. Phosphate Mixtures, (a) One-fifteenth 
Molecular Acid or Primary Potassium Phospihate (KH0PO4).— The 
pure re-crystallized salt should be used. 9.078 gm. are dissolved in 
freshly distilled water. To this are added 200 cc. of 0.01 per cent, 
aqueous solution phenolsulphonephthalein and the volume is brought 
up to one liter with distilled water. { 

(6) One-fifteenth Molecular Secondary Alkaline Sodium Phosphate. 
—The pure, recrystallized salt (NacHFOj.HiO) is exposed to the 
air, protected from dust, from ten days to two weeks. Ten mole- 
cules of water are given off and a salt of this formula is obtained, 
Xa-jP0 4 .2H 2 0. Of this salt, 11.S7G gm. are dissolved in distilled water, 
200 cc. of 0.01 per cent, aqueous solution phenolsulphonephthalein 
are added, and the whole made up to one liter with distilled water. 
The same amount of indicator must be added to the two solutions and 
to the salt solution which is hereinafter described. Mixtures are 
made according to the amounts given in ccs. in the table below to 
give differing hydrogen-ion concentrations. After the solutions have 
been prepared, a crystal of thymol is placed in each flask to prevent the 
growth of moulds. Small test-tubes about 8 mm. bore by 100 mm. 
are partially filled with the solutions. The glass should be of a type 
which does not give off alkali readily, such as Non-sol, Pyrex, or Jena. 
The upper ends of the tubes should be sealed in the blow-pipe. They 



TESTS FOR ACID INTOXICATION 



209 



should be kept in a dark place, under which condition they retain 
their colors for some time. 



PKOPORTIONS IN WHICH PHOSPHATE SOLUTIONS ARE MIXED. 



Hydrogen-ion concentration 



Primary potassium phosphate, cc. 
Secondary sodium phosphate, cc. 



7.0 


7.2 


7.4 


7.6 


7.8 


8.0 


8.2 


8.4 


37.0 
63.0 


27.0 
73 


19.0 
81.0 


13.2 
86.8 


8.8 
91.2 


5.6 
94.4 


3.2 
96.8 


2.0 
98.0 



1.0 
19.0 



The terminology which is employed in discussing the question of acidosis is confusing 
to one unaccustomed to the physico-chemical methods of expression. We can do no 
better than to quote verbatim the concise explanation given by Levy, Rowntree and 
Marriott: "A solutionis acid when it contains an excess of hydrogen over hydroxyl 
ions, neutral when hydrogen and hydroxyl ions are in equal numbers, and alkaline 
when hydroxyl ions predominate. An acid of 'normal' strength contains, in 1 liter, 
1 gram of hydrogen capable of forming ions, and its strength may be expressed as 1 N. 
Diluting such a solution ten times, we would have a ^ N or a solution containing ^ 
gram of actual or potential hydrogen ions to the liter. Continuing the process of 
dilution until TrnTTnnnnr normal acid is obtained, we would have in such a solution 
TffoTfWo o § ram °f hydrogen ions. Pure water, however, dissociates to form hydrogen 
ions, and at 20° C. contains approximately toWttotto oi hydrogen ions to the liter and 
an equivalent amount of hydroxyl ions. That is to say, pure water, our standard of 
neutrality, is xotjotuo o N acid and also t^oto o oxr alkaline. To avoid writing large 
figures it is customary to use the logarithmic notation and to express t (To To oo"o~ N, as 
10- 7 N or, more conveniently, as suggested by Sorensen, to drop the 10 and the minus 
sign and say pH7. If we have less than yrnnrVcnnr g ram of hydrogen ions to the liter, 
the solution is less acid than water, that is, is alkaline — so, pH8 means actually 
ToWcnnnr N alkali. The higher the exponent the more alkaline, or what is saying the 
same thing, the less acid is the solution. 
To sum up: 

pHl = N/10 acid. 

pH6 = N/1,000,000 acid. 

pH7 = Neutrality. 

pH8 = N/1,000,000 alkali. 

pH14 = N/10 alkali. 

The reaction of the blood serum varies approximately between pH7 and pH8, the 
neutral point being reached only in severe uncompensated acidosis, and a reaction of 
pH8 being attained perhaps only after the administration of alkalies." 



Preparation of Sacks.— Anthony's negative cotton is used. This is 
manufactured by the Ansco Company, Birmingham, N. Y. It con- 
tains about 30 per cent, of water. This must be poured off and the 
celloidin rinsed in two or three changes of 95 per cent, alcohol before 
being dissolved. One ounce is then dissolved in 500 cc. of a mixture 
of equal parts of ether and 95 per cent, alcohol. The solution should 
be allowed to stand for a week to permit the impurities to settle. 

In making the sacks, a small test-tube about 50 mm. long by 6 mm. 
bore with a flared mouth is filled with the celloidin solution. It is 
poured out of the tube slowly, rotating the tube so that the inside will 
be coated with a thin film of celloidin. The tube is then held in the 
upright condition so that it may drain for ten minutes, or until it ceases 
to give off the odor of ether. When the sacks are dried too long they 
14 



210 EXAMINATION OF THE BLOOD 

become hard and brittle, while sacks that have not been dried long 
enough become white and cloudy when water is added. 

When the sacks have dried for a suitable length of time they are 
filled with water and removed from the tubes by running a knife blade 
around the edge at the top of the test-tube to loosen them, running 
water between the sack and the tube. The sacks are preserved in 
water. 

Preparation of Salt Solution. — Eight gm. of chemically pure sodium 
chloride are dissolved in a small quantity of distilled water. 220 cc. of 
0.01 per cent, aqueous solution of phenolsulphonephthalein are added 
and then enough water to bring the total volume to one liter. 
Marriott recommends making the concentration of the indicator 
stronger in the salt solution than in the standard phosphate mixtures 
to allow for loss during the process of dialysis. It is important that the 
solution contains no free alkali or any acid except carbonic acid. A 
portion of the solution should be tested by boiling it in a Pyrex glass 
test-tube to expel carbonic acid. This is cooled quickly and compared 
with the standards. It should match the pH7 tube. Should the glass 
of the test-tube give off alkali, the solution will be colored a faint pink. 
Should the reaction of the solution differ from this, it may be corrected 
by adding a few drops of very dilute acid or alkali to the entire bulk of 
the solution. The salt solution must be kept in a flask of Non-sol or 
Pyrex glass, or in a flask of ordinary glass whose inner surface has been 
coated with a thin film of paraffin.* 

Procedure for the Determination.— The procedure should be carried 
out in a room free from acid or ammonia vapors. While either sepa- 
rated serum, the plasma from oxalated blood, or whole blood may be 
employed, serum separated from the clot is to be preferred since the 
introduction of oxalate may introduce an error. The serum should be 
separated by centrifugalization as soon as possible after the blood has 
been obtained. Hemolysis should be guarded against. 

One-half cc. of clear serum is transferred with the pipette to one of 
the sacks, which should have been washed inside and out with the salt 
solution. In a test-tube about 8 mm. in bore and 50 mm. long is 
placed 2 cc. of the salt solution containing the indicator. The level 
of the fluid on the inside of the sack should not exceed that on the 
outside. After seven minutes have elapsed the sack is withdrawn and 
the dialysate (salt solution) is transferred to a test-tube 100 to 140 mm. 
long, having the same bore as those containing the standard phosphate 
solutions. The atomizer bulb is connected with a piece of glass tubing 
the end of which has been drawn out into a fine capillary. The capil- 
lary end is placed in the tube of dialysate and a current of air is blown 
through to get rid of carbon dioxide. The force of the air-current 
should not be strong enough to cause the liquid to bubble out of the 
tube. After the air-current has been allowed to pass for three minutes, 
the color of the tubes is compared with that of the standards. When 
the unknown does not exactly match either of two tubes but appears 



TESTS FOR ACID INTOXICATION 211 

to fall between them in depth of color, the reading should be interpo- 
lated. 

Interpretation of Results.— Marriott states that with normal indi- 
viduals the alkali reserve (R) was found to be between 8.55 and 8.45, 
when the subjects were on a mixed diet. After a fast of sixteen hours, 
a normal adult's serum gave a reading of 8.35. The sera from infants 
gave somewhat lower readings, a value of 8.3 being encountered 
occasionally. He felt that values of 8.4 to 8.5 corresponded to alveolar 
carbon dioxide tensions of 38 to 45 mm. Values between 8.0 and 
8.3 correspond to alveolar carbon dioxide tensions of 28 to 35 mm. and 
indicate a moderate degree of acidosis, while values as low as 7.7, 
corresponding to an alveolar carbon dioxide tension of 20 mm. were 
found in severe acidosis and indicated that the individual was in grave 
danger. 

Significance of the Results of Blood Tests for Acidosis.— Two tests have 
been applied to the blood for the detection of acidosis : 

1. Determination of the carbonate content of the whole blood or 
plasma— the so-called alkaline reserve. 

2. Determination of the hydrogen-ion concentration (pH.) of blood. 
The latter merits little consideration because the results obtained by its 
use show little or no deviation from the normal except in moribund 
cases. Indeed it appears very likely from animal experimentation 
and from theoretical considerations that any sustained elevation in the 
actual (pH) of the blood is incompatible with life. 

Since the bicarbonate of the body fluids represents the most import- 
ant alkaline reserve of the tissues, it is apparent that this first line of 
defence as it were will show deviations from the normal, in conditions 
of acidosis. It is well established that the blood bicarbonate is depleted 
in acidotic conditions caused by fixed acids. As examples we may 
refer to the production of unoxidized fatty acids, like 0-oxybutyric and 
aceto-acetic in diabetes, and the improper elimination of acids produced 
in normal metabolism as happens in some cases of nephritis. 1 

When, however, an accumulation of carbonic acid occurs in the 
body the bicarbonate of the blood is elevated above the normal. This 
has been demonstrated by R . W. Scott in animals made to breathe C0 2 
and recently he has reported a similar condition in patients with 
chronic pulmonary emphysema of the "large lunged type." 2 

Determination of Chlorides of Blood.— The methods which are given 
may be used for determining the chloride content of either plasma or 
whole blood. The content of whole blood normally amounts to 0.45 
to 0.50 per cent.; figures for the plasma are about 0.12 per cent, higher. 
While it would seem more logical to determine the content of the 
plasma, which bathes the body tissues, the examination of whole 
blood is really preferable, since unless the plasma is separated from the 

1 A recent discussion of acidosis in nephritis with bibliography is given by Chase and 
Meyers, Jour. Am. Med. Assn., 1920, lxxiv, 641. 

2 Proc. Soc. Exp. Biol, and Med., 1919, xvii, p. 18. 



212 EXAMINATION OF THE BLOOD 

cells almost immediately, its chlorides increase at the expense of the 
corpuscles due to passage of carbon dioxide from plasma to the cor- 
puscles or its escape into the air. 

Discussion of -Methods.— The two methods given are equally accu- 
rate. The method of Myers and Short has certain points of advantage 
over Austin and Van Slyke's iodometric method. The reagents are 
dilutions of those used for the determination of urinary chlorides by 
the Volhard-Harvey method ; they are more stable ; and the procedures 
are carried out more rapidly. Furthermore, if methods be used for 
determining blood sugar and creatinine which employ picric acid to 
precipitate proteins, a portion of the same protein-free filtrate may be 
used for the chloride determination. 

Method of Myers and Short.— Principle.— The proteins are pre- 
cipitated with picric acid as in the methods for the determination of 
blood sugar and creatinine. After separation of the precipitate, the 
protein-free filtrate is used for the determination of chlorides by the 
Volharcl-Harvey method, the principle of which is described in the 
method for determination of urinary chlorides. 

Reagents Required.— 

1. Silver nitrate solution, 2.904 gm. to 1000 cc. of water. One cc. 
is equivalent to 1 mg. of sodium chloride. This may be prepared by 
making a one to ten dilution of the solution used by determining urinary 
chlorides by the Volhard-Harvey method. 

2. Acid ferric alum indicator solution prepared by dissolving 100 gm. 
of crystalline ferric ammonium sulphate in 100 cc. of 25 per cent, nitric 
acid and adding 4 volumes of distilled water. This is one-fifth the 
strength of the solution used for the urine determination. 

3. Ammonium thiocyanate of such strength that 2 cc. are equivalent 
to 1 cc. of the silver solution (No. 1). It contains approximately 0.65 
gm. of the thiocyanate in 1000 cc, but must be checked by titration 
against the silver nitrate solution. 

4. Picric acid crystals, C. P. 

Procedure.— In a 50 cc. centrifuge tube are placed 3 cc. of whole 
blood (or plasma) and 27 cc. of distilled water. About 0.5 gm. of dry 
picric acid are added and the mixture is stirred until protein precipita- 
tion is complete, when a bright yellow "mustard" color appears. The 
tube is centrifugalized and the supernatant clear fluid is decanted into a 
dry beaker. Should any particles remain the filtrate is poured through 
a dry filter. 20 cc. of the filtrate are transferred with a pipette to a 
clean dry centrifuge tube when 20 cc. of the standard silver nitrate 
solution and 10 cc. of the acid ferric alum indicator are added. After 
stirring, the precipitated silver chloride is thrown down by centri- 
fugalization and the clear supernatant fluid decanted into a dry 
beaker. Duplicate portions of 20 cc. each are measured into porcelain 
evaporating dishes with pipettes. Titration is carried out by adding 
the ammonium thiocyanate solution until the first permanent tinge of 
brown is obtained. While the end-point is definite, some experience 



TESTS FOR ACID INTOXICATION 213 

may be required before it is recognized invariably. Passing the end- 
point by one drop introduces an error of 0.5 per cent, in estimating the 
chlorides in 100 cc. of blood. 
- If desired, the 1 : 5 picric acid filtrate may be used for sugar and 
creatinine. In this case 5 cc. (the equivalent of 1 cc. of blood) are 
taken adding 10 cc. of the standard silver solution and 10 cc. of a 
diluted acid ferric alum indicator solution (the solution described above 
as No. 2 diluted with an equal quantity of water). After precipita- 
tion of the chlorides and centrifugalization, a single 20 cc. portion of 
filtrate is titrated as described above. Myers and Short have shown 
that the results above with 1:5 and 1:10 filtrates are practically 
identical. 

Calculation.— This is made with the formula: 

10— I — — X ~\ X 100 = mg. of sodium chloride in 1000 cc. of blood (or plasma). 

Austin and Van Slyke's Method for Determining Blood Chlorides.— 
Principle.— The proteins are precipitated from blood serum or an 
oxalated specimen of whole blood with picric acid. The coagulum is 
removed and the chlorides in the protein-free filtrate are precipitated 
in the presence of nitric acid by a standard silver nitrate solution. The 
amount of uncombined silver nitrate is determined by titration with 
a standard potassium iodide solution in the presence of nitrous acid and 
starch. The first drop of iodine in excess of the silver present is changed 
to free iodine and gives the blue starch-iodine color reaction. The 
optimum acidity for the end-point is secured by the addition to the 
starch solution of trisodium citrate. Knowing the uncombined silver, 
the combined silver is readily found, and the amount of chlorides 
determined from this. 

Reagents Required.— 

Solution 1.— An acid solution of silver nitrate, 1 cc. of which is equiva- 
lent to 2 mg. of sodium chloride. 

Silver nitrate 5.812 gm. 

Nitric acid (sp. gr. 1.42; 250.000 cc. 

Distilled water to 1000.000 cc. 

McLean and Van Slyke state that in place of silver nitrate, metallic 
silver may be employed. This may be obtained in a high state of 
purity. In this case, 3.688 gm. of silver are dissolved in the prescribed 
amount of nitric acid and the total volume brought to 1000 cc. with 
distilled water. It may be convenient to make up a stock solution of 
silver nitrate of ten-fold this concentration and to prepare the test 
solution from this by dilution. The strength of the solution may be 
checked by titration against a known solution of sodium chloride, as 
directed on page 250. 

Solution 2 (Van Slyke and Donleavy).— A solution of potassium 
iodide, 1 cc. of which is equivalent to 0.8'mg. of sodium chloride. 

Potassium iodide 2.4 gm. 

Distilled water to 1000.0 cc. 



214 EXAMINATION OF THE BLOOD 

This solution must be standardized against the silver solution and 
diluted to the extent indicated by the preliminary titration. Five cc. 
of the silver solution (1) measured with a calibrated pipette are added 
to 5 cc. of the starch solution (3) and 5 cc. of water. The iodine 
solution is run in from a burette until the blue end-point is obtained. 
It should require exactly 12.65 cc. of the iodine solution to secure this 
end-point with 5 cc. of the silver solution, 12.50 being required to 
precipitate the silver and 0.15 cc. additional to give the end-point. 
The iodine solution must be diluted to secure this concentration. 

Solution 3.— This is a solution for use in the final titration containing 
sodium citrate, sodium nitrite, and starch, which substances respec- 
tively regulate the acidity, provide an oxidizing agent for the iodine, 
and serve as an indicator. 

Sodium citrate, crystalline (Na3C 6 H 5 07.5|H20) . . . . 446.0 gm. 
Sodium nitrite (NaN0 2 ) 20.0 " 



Soluble starch 



2.5 



Distilled water to 1000.0 cc. 

The starch should be dissolved by boiling in about 500 cc. of water. 
The citrate and nitrite are then added, heating the mixture until all 
are dissolved. While still hot the solution is filtered through cotton, 
the filter washed with hot water; the filtrate allowed to cool; and 
the volume made up to 1000 cc. Filtration serves to remove 
insoluble substances, chiefly occurring in the nitrite. The solution 
keeps indefinitely, and though it becomes cloudy, its efficacy is not 
impaired. 

Procedure— In a 60 cc. volumetric flask are placed 3 cc. blood-serum 
or whole blood with 15 cc. of water. The pipette is rinsed by drawing 
up and ejecting some of the water two or three times into the pipette. 
Only a pipette should be used which has been carefully calibrated. 
Thirty cc. of a saturated solution of picric acid should be added and 
sufficient water to bring the total volume to 60 cc. When a volumetric 
flask is not available, the water (27 cc. in all) and the picric acid solution 
may be added from burettes. The contents of the flask are mixed and 
filtered after ten minutes' standing. To 40 cc. of the filtrate are added 
10 cc. of the standard silver solution (Solution 1) with two drops of 
caprylic alcohol. The solution should be thoroughly mixed and 
allowed to stand over night when the clear supernatant fluid is decanted 
through a small filter paper. Duplicate portions of the filtrate of 20 cc. 
each are placed in 100 cc. Erlenmyer flasks with a calibrated pipette. 
Four cc. of the starch-citrate solution (Solution 3) are added to each 
flask, and the standard iodide solution (Solution 2) is run in from a 
burette until a permanent blue end-point is obtained. Only a per- 
manent and unmistakable blue is taken as the end-point. 

Calculation- The formula for calculation has been simplified by 
Van Slyke and Donleavy to the following: 

Mg. of sodium chloride per cc. of blood, or \ _ 1Q lfi _ cc . KI solution. 
Gm. of sodium chloride per liter of blood J 



TESTS FOR ACID INTOXICATION 215 

In other words, the number of cc. of IvI solution used to secure the 
end-point is subtracted from 10.15 and the result represents the number 
of mg. of sodium chloride per cc. of blood. The figure 10.15 is used 
instead of 10 because of the fact that an excess of 0.15 cc. of iodine 
solution is necessary to produce the end-point. 

Caution.— Attention is directed by Van Slyke and Donleavy to the 
necessity for calibrating all pipettes and volumetric glassware used in 
this determination, because there is not much difference between the 
significant figure and the normal figure of blood chlorides. This may 
be done by weighing at a temperature of 20° C. the pipettes, etc., dry 
and when filled with water, and by noting the difference in the weights. 

Findings.— Normally, the chloride content of whole blood is from 
0.45 to 0.50 per cent, and of plasma from 0.57 to 0.62 per cent. High 
chloride contents have been found in nephritis, especially in paren- 
chymatous nephritis, certain cardiac conditions, anemia, and in some 
cases of malignancy, while low values are observed in fevers, diabetes, 
and pneumonia. The excretion of chlorides and nitrogen appear to be 
more or less independent functions, since in parenchymatous nephritis 
the excretion of chlorides is much more impaired than that of nitrogen 
while with interstitial nephritis, the reverse is true. 

Determination of Oxygen of the Blood.— The oxygen content of blood 
is of significance in certain clinical conditions, notably in cardiac 
decompensation. It is stated that in normal individuals, the oxygen 
content of venous blood is about 13.5 volume per cent., or two-thirds 
of the total amount in the blood when it is saturated. Lundsgaard 
recommends the following method for determination, involving two 
steps, first, the drawing of a sample of venous blood, and second, the 
determination of its oxygen content. Oxygen is determined by 
Van Slyke's technic, which has been described previously (see page 20). 
The apparatus for drawing blood consists of a very sharp needle con- 
nected by means of a piece of rubber tubing 3 or 4 cm. long to a glass 
pipette of about 0.5 cm. bore. The pipette and rubber tubing should 
contain sodium oxalate crystals. These are introduced by wetting 
the interior with a saturated solution of oxalate, which is dried with a 
current of air. The upper end of the pipette is stuffed with cotton 
and placed in the mouth when the needle is thrust into the vein in the 
proximal direction. Only gentle suction will be needed in drawing the 
blood. Should air be aspirated through the blood, the specimen should 
be discarded and a new pipette used, keeping the needle and rubber 
connection in situ. When from 6 to 12 cc. of blood have been secured, 
the upper end of the pipette is closed with the finger and the needle 
is withdrawn. The rubber tubing is disconnected and the blood is 
discharged into a cylinder of about 2 cm. diameter (with a few crystals 
of sodium oxalate in the bottom) under a layer of white mineral oil 
about 2 cm. deep. The last 0.5 or 1.0 cc. of blood is discarded on 
account of oxidation. The blood in the cylinder is then mixed with the 
oxalate by stirring with a glass rod. From the cylinder, 2 cc. samples 



216 EXAMINATION OF THE BLOOD 

are introduced into Van Slyke's apparatus beneath a layer of evacuated 
dilute ammonia. The blood should be stirred before removing each 
sample to secure homogeneity. Only fresh specimens should be used. 
The oxygen content and the oxygen combining power are determined. 
The latter may be calculated from the determination of hemoglobin 
by Palmer's method. The difference may be found between the 
oxygen in venous blood and the total oxygen combining power of the 
hemoglobin. This difference is termed oxygen vnsaturation of the 
venous blood by Lundsgaard, who found that in normal individuals 
the values ranged from 2.5 to 8 volume per cent., while with uncom- 
pensated heart disease, the values were from 9.7 to 15.2 volume per 
cent. Since normal figures were found only in a stage of full com- 
pensation or in decompensation with rapidly lessening symptoms while 
figures above the upper normal extreme were met with (1) during 
decompensation, (2) during compensation just before clinical symp- 
toms of decompensations developed, or (3) at times in patients with 
auricular fibrillations in complete and stable compensation, he feels 
that the determination of oxygen unsaturation afforded an objective 
criterion of the positive effect of digitalis therapy. 



CHAPTER II. 
EXAMINATION OF URINE. 

Introductory.— The examination of urine yields much information 
of great value to the clinician. The writer has often felt, however, that 
the question of urinary examinations has been more strongly empha- 
sized by many writers and by occasional clinicians than was justified 
by the value of the information received. Certain tests are described 
and carried out whose diagnostic value is questionable and much of the 
quantitative work which is done is valueless, even from the standpoint 
of routine records, because no attention has been paid to the intake of 
protein, carbohydrates, and chlorides. Indeed the use of quantitative 
methods in urinalysis is irrational unless at least the twenty-four-hour 
output be obtained (a simple consideration frequently overlooked), and 
unless the diet be taken into consideration. The examination of the 
urine for clinical purposes can be and should be reduced to extremely 
simple terms. An analysis of urine should be part of the routine 
examination of the patient and may be made by the clinician or a 
trained assistant with modest equipment and small expenditure of 
time. When indicated, bacteriological examinations and quantitative 
determinations may be referred to a well-equipped laboratory, though 
there is no reason why a clinician with a modern training should not 
be able to carry out readily any useful urinary examination. 

In this section, we shall attempt to give an outline for a simple routine 
examination of the urine, to indicate tests of real clinical significance, 
to recount the clinical significance of the findings, and to describe the 
technic of quantitative tests which may be desired in exceptional cases. 

ROUTINE EXAMINATION. 

A routine examination of the urine should include the following : 

1. Color. 

2. Appearance. 

3. Reaction. 

4. Specific gravity. 

5. Qualitative test for albumin. 

6. Qualitative test for sugar. 

7. Microscopical examination of sediment. 

In case quantitative determinations are desired, a twenty-four-hour 
specimen should be obtained. 

Apparatus Required.— The laboratory equipment which is needed for 
the routine examination of the urine is included in the minimal equip- 
ment for the clinical laboratory. (See Appendix.) 



218 EXAMINATION OF URINE 

Daily Amount.— It should be remembered that a knowledge of the 
amount excreted in twenty-four hours is of the greatest importance in 
the clinical management of the various forms of nephritis, cardiac 
decompensation, and diabetes mellitus. This simple observation, 
judiciously interpreted, may give vastly greater information than 
laborious quantitative determinations of various constituents. When 
there is a complete suppression of urine, the condition is spoken of as 
anuria or anuresis; when the urine is greatly diminished in amount, 
the term oliguria or oliguresis is applied. Polyuria refers to an in- 
creased quantity. 

Order of Procedure.— In order to conserve a specimen when only a 
small quantity is furnished, a definite line of procedure should be 
followed. It is possible to make a complete qualitative examination 
with 50 cc. or even less if economy be exercised. First, take the 
specific gravity, then start a portion in the centrifuge, next take the 
reaction, perform the test for albumin and sugar, and finally examine 
the sediment microscopically. It is better to know one or two tests 
thoroughly and to know their limitations than to have a superficial 
acquaintance with many methods. The following are recommended 
because of their simplicity and their reliability. 

Appearance of Specimen.— This should give a clue to special tests. 
A dark brown color suggests the presence of bile; reddish or chocolate- 
colored urine, the desirability of chemical and microscopical tests for 
blood, though scanty and febrile urines are dark because of concentra- 
tion. Turbidity may be due to (1) bacteria, in which case no clearing 
will be seen on heating or by the addition of acid; (2) phosphates which 
dissolve on the addition of acids; (3) urates, which may be white or 
red. In the latter instance the color may deceive the patient into 
thinking that blood is present, but cloudiness disappears promptly 
on heating; (4) pus, which may be centrifugalized to the bottom of the 
tube but dissolves neither with heat nor with dilute acid and is, of 
course, readily recognized on microscopic examination. 

Mucous shreds should be noted when present. They are white 
bodies which are readily visible macroscopically, looking like small 
curled threads, occasionally assuming an almost spiral form. AYhen 
teased out upon a slide and stained with methylene blue they will be 
found to consist of an albuminous matrix with embedded epithelial 
cells, polymorphonuclear leukocytes and bacteria, and a variable 
amount of mucus. Frequently gonococci may be demonstrated, since 
they occasionally persist for years in the urine of apparently cured 
cases. They are sometimes referred to as "gonorrheal shreds" or 
"clap shreds." 

Preservation of Specimens. -If possible the specimen should be 
examined shortly after it has been voided. When prompt examination 
is impossible and it is necessary to keep the specimen, a preservative 
should be added, such as 2 drops of 40 per cent, formalin to the liter of 
urine. In making chemical analysis, however, it should be remembered 
that formalin may reduce Fehling's solution. 



ROUTINE EXAMINATION 



219 



Specific Gravity.— The float (Fig. 61, a) should rest in the cylinder of 
urine without touching the sides of the cylinder. The reading should 
be taken from the bottom of the meniscus. If foam is bothersome, a 
drop or so of ether may be added to cause its subsidence. A urinom- 
eter has been placed on the market recently which makes use of the 




Fig. 61. — Apparatus used in routine urinalysis, a, urinometer ; b, new type of urinometer; 
c, conical sediment glass; d, centrifuge tube. 



principle of the hydrometer commonly used in testing storage batteries 
(Fig. 61, b). With this the urine may be aspirated into a cylinder 
containing the hydrometer float. It should prove especially useful 
where many specimens are to be examined, as in a hospital laboratory. 
It is needless to say that it should be rinsed carefully before and after 
each test. 



220 EXAMINATION OF URINE 

Qualitative Tests for Protein Bodies. -The following tests react with 
both albumin and globulin, which we do not attempt to consider 
separately in clinical work. These tests are preferred because of their 
simplicity and because of the ease with which the reagents can be 
obtained. The test with heat and acetic acid is the more delicate, 
sufficiently so for all clinical purposes. We do not feel that more 
delicate tests than these are either necessary or desirable, since the 
extremely faint traces of albumin which they reveal are of doubtful 
clinical significance. 

Heat and Acetic Acid Test-Fill a clean test-tube almost to the top 
with clear urine. If turbid the urine should be filtered, or the super- 
natant clear urine in the centrifuge tube may be used. When clear 
urine can be obtained' in neither way, a small amount of rvieselguhr 
should be shaken up with the urine, which may then be filtered. Heat 
the top layer in the test-tube to the boiling point. The bottom portion, 
should not be heated since it serves as a contrast. If a cloud occur 
add a few drops of 5 per cent, acetic acid. Stronger acid or an excess 
of acid should be avoided, since either will cause the albumin to go into 
solution. This urine is heated again. If the cloudiness remain alter 
the addition of acid, it is due to albumin or nucleoprotein, but it it 
disappear, it is due to phosphates. The reason for the appearance of a 
precipitate of phosphates when previously clear urines are heated is 
that carbon dioxide is liberated by heating, and the reaction ol the 
urine is made more alkaline. - . • -j ,n t> \ 

Heller's Nitric Acid Ring Test.-Vut about 3 cc. of nitric acid (C. r\) 
in the bottom of a clean test-tube and allow 5 cc. layer of urine to run 
over the acid from a pipette. This should be done slowly and care- 
fully so that there will be distinct layering of the two fluids and so that 
mixing does not occur (Fig. 62). A white layer at the junction indi- 
cates the presence of albumin. A similar layer may be given by certain 
drugs. A white ring is occasionally given by uric acid or the urates. 
This ring usually has a more diffuse upper border and occurs at times 
above the line of contact. As a confirmatory measure the urme may be 
diluted with 3 or 4 parts of water and the test repeated with nitric acid. 
Uric acid or the urate will not give a ring when the urme has been so 
diluted. , . , i 

Sulphosalicylic Acid Test.-The urine is placed in a test-tube and a 
drop or so of a 20 per cent, solution of sulphosalicylic acid is added. 
A white precipitate is seen when albumin is present. The urine may 
be layered over a small quantity of the acid as was directed lor Heller s 
test. This reagent does not give a precipitate with the resins or with 
uric acid. Albumose gives a precipitate which clears up on heating 
and reappears on cooling. . . , 

Magnesium Sulphate Test.-Vut 3 or 4 cc. of acid-magnesium sul- 
phate mixture in a test-tube and run the urine from a pipette to lorm 
a layer over the reagent. A white ring will be formed at the junction 



ROUTINE EXAMINATION 



221 



when albumin is present. The reagent is prepared by adding 1 part 
of nitric acid (C. P.) to 3 parts of a saturated solution of magnesium 
sulphate. 




-Urine 
•Nitric Acid 

Fig. 62. — Heller's nitric acid test for albumin. 



222 EXAMINATION OF URINE 

Record of Albumin Findings.— In recording findings, it is advisable 
to adopt a fixed terminology for the sake of comparison. The following 
is suggested. It is based on the density of the cloud obtained with the 
heat and acetic acid test. FPT = faintest possible trace, meaning the 
least cloud which is visible when the tube is held against a dark back- 
ground. FT=faint trace. The cloud is slight but readily visible 
without a dark background. T=trace. There is a distinct cloud, 
which is sufficiently dense to render the tube not transparent, til - 
heavy trace. The cloud is dense and shows flakes. In such cases, 
albumin is present in a measurable quantity. 

Significance of Albuminuria. -In normal individuals, plasma proteins 
do not escape into the urine in quantities which would render them 
appreciable by the methods usually employed. They may filter 
through under certain abnormal conditions due to change in the 
permeability of the kidney brought about by inflammation or asphyxia, 
the condition being referred to as renal albuminuria. In addition, 
protein may be found in the urine without renal damage due to the 
admixture of inflammatory exudates in other parts of the urinary tract, 
the so-called accidental albuminurias. 

Dietetic albuminurias are recognized and are seen after the ingestion 
of abnormally large amounts of protein. 

Renal albuminuria is occasionally seen in normal persons alter 
mental strain, severe bodily exertion, cold baths, and in cases of so- 
called functional albuminuria, especially in lordotic or orthostatic 
albuminuria. Such cases are always to be regarded with suspicion and 
should not be dismissed until carefully studied to ascertain the under- 
lying cause. Even when albuminuria is apparently functional, in 
occasional cases definite evidence of kidney damage has been found 
later, pointing to the fact that so-called physiological albuminuria 
occurs only where there is a slight insufficiency of the kidney. 

Albuminuria of renal origin may be seen in the anemias and leuk- 
emias, in chronic passive congestion of the kidneys, as in uncompen- 
sated heart disease, in febrile conditions, in poisoning by certain drugs, 
notably by ether, mercury, cantharides, lead, arsenic, etc. In these 
cases, the kidney tissue may be damaged, either permanently or 
temporarily, or the lesion may be a simple passive congestion, and. may 
clear up when the underlying cause is removed. The true nephntides 
give varying findings. In acute nephritis albumin is usually present 
in large amount, and diminishes as the inflammatory process subsides. 
In chronic nephritis of the interstitial or arteriosclerotic type, the 
amount is frequently small and repeated examinations may be neces- 
sary to demonstrate even faint traces, whereas with so-called paren- 
chymatous nephritis, the amount is often much larger. In this 
particular, however, it is very difficult to generalize, since the classifica- 
tion of the forms of nephritis on clinical grounds is none too sound at 
best. 



ROUTINE EXAMINATION 223 

Accidental albuminuria may be seen whenever there is opportunity 
for the admixture of blood or of inflammatory exudates with the urine 
in the urinary tract below the kidney proper. It may be due to 
menstrual blood, or blood from hemorrhage from trauma or new growth, 
to pus from pyelitis, ureteritis, cystitis, urethritis, vaginitis, or to 
addition of lymph, spermatic or prostatic fluid. Before drawing con- 
clusions as to the existence of nephritis, due care must be taken to 
exclude any of these conditions as possible sources of error. 

Qualitative Tests for Sugar.— Benedict's Test.— This is much to be 
preferred, not only on account of its delicacy but because of the per- 
manency of the reagent. 

The formula for Benedict's solution is: 

Copper sulphate 17.3 gm. 

Sodium citrate 173.0 " 

Sodium carbonate (anhydrous) 100.0 " 

Distilled water, to make 1000 cc. 

The sodium citrate and the sodium carbonate are dissolved by heat- 
ing with 600 cc. of water, after which the resulting solution is filtered 
through a folded filtrate into a graduate and the total volume is made 
up to 850 cc. with water. The copper sulphate is dissolved separately 
in 100 cc. of water, when the bulk of this is made up to 150 cc. The 
sodium carbonate and citrate solution is now poured into a large 
beaker and the copper sulphate is added slowly with continual stirring. 

Procedure.— In a test-tube place 5 cc. of the reagent and add not 
more than 8 to 10 drops of the urine. Boil the mixture thoroughly 
and allow to cool spontaneously. If glucose be present, the entire 
body of the solution will show a precipitate, ranging from green to red 
in color, according to the sugar content of the urine. In the absence of 
sugar, the solution remains quite clear or shows only a faint bluish 
turbidity. 

Fehling's Test. — The formulae for the two Fehling's solutions are: 

Solution No. 1. Copper Sulphate Solution. 

Copper sulphate 34.65 gm. 

Distilled water, to make 500.00 cc. 

Solution No. 2. Alkaline Tartrate Solution. 

Potassium hydroxide 125.00 gm. 

Rochelle salts 173.00 " 

Water, to make . 500.00 cc. 

The solutions are kept in separate bottles, and small portions as 
needed are prepared by mixing equal quantities. 

Procedure. — Fehling's solution should be prepared fresh daily, by 
mixing equal quantities of the two solutions. After the mixture has 
been made, boil a little in a test-tube to make sure that no reduction 
occurs. 



224 



EXAMINATION OF URINE 



To about 5 cc. of hot Fehling's solution add a few drops of urine, 
boil, and continue adding urine, a few drops at a time, until there are 
equal quantities of urine and Fehling's solution. Sugar is indicated 
by yellow or red precipitate. The appearance of a greenish color 
means nothing. In case of doubt, allow the tube to stand. When 
sugar is present, a distinct precipitate settles to the bottom of the tube. 

Fermentation Test— A doubtful result with either of the foregoing 
reduction tests should be confirmed by a fermentation test, since 
substances other than glucose, such as uric acid, formalin, creatinine, 
chloroform and simple aldehydes may give a slight reaction. 



1 



— TJ 



7 

a 

5" 



iLO 




Fig. 63. — A, Esbach albuminometer; B, Einhorn saccharometer; C, improvised 
fermentation tube. 



Two fermentation tubes (Fig. 63, B) are employed. In one the urine 
to be examined is placed after it has been thoroughly mixed with about 
one-sixteenth of a fresh cake of compressed yeast. The second tube is 
filled with normal urine, similarly mixed with an equal amount of yeast. 
The two tubes are kept at room temperature or may be placed in the 
incubator. The formation of gas in the upright denotes the presence 
of glucose providing the normal urine shows no gas formation. 

In the absence of fermentation tubes, ordinary test-tubes may be 
employed, filling the tube with the urine and yeast mixture, and then 
setting it upright, with the opening down immersed in a beaker of the 
same urine. (Fig. 63, C). 

Phenylhydrazine Reaction.— This reaction may be used to control 
the results of the reduction tests and has the advantage over the 



ROUTINE EXAMINATION 225 

fermentation test that the results may be obtained in much shorter 
time. The reaction depends upon the formation of an osazone, which 
may be identified by its characteristic crystals. The test is an excellent 
one, since there are practically no substances except the carbohydrates 
which gave the reaction. Levulose and dextrose give identical osa- 
zones. The osazone formed by maltose can be identified by its 
crystals, and lactose will not give the crystals when the test is carried 
out as directed. 

Kowarsky's method is preferred for clinical work. Five drops of 
the phenylhydrazine (the base) are mixed in a test-tube with 10 drops 
of acetic acid and 1 cc. of a saturated solution of sodium chloride. 
A curdy mass results, when 2 or 3 cc. of the urine to be examined and 
4 or 5 cc. of water are added. The mixture is heated for about two 
minutes over the flame. Care should be taken to avoid bumping. 
This may be accomplished by constant shaking or by putting some 
short bits of glass tubing in the test-tube. When glass tubing is used, 
the fluid should be poured into another test-tube to cool. Frequently 
the crystals appear rapidly. If they do not, at least thirty minutes 
should be given, since this time may be required for crystallization 
when the amount of sugar is small. The deposit should be removed 
with a pipette, placed upon an object slide, and examined under the 
low power of the microscope. The crystals are yellow and needle- 
shaped, grouped in clusters or sheaves. 

Significance of Glycosuria. — Normally, the urine contains very min- 
ute quantities of sugar, but not enough to be detected by the ordinary 
qualitative tests. On this account, Benedict has suggested using the 
term glycuresis instead of glycosuria. The normal glucose content of 
the circulating blood is about 0.1 per cent. When the concentration 
rises to 0.2 per cent, or thereabouts, constituting a hyperglycemia, 
glucose is secreted by the kidney into the urine. The occurrence of 
hyperglycemia and the resultant glycosuria may be due to defective 
storage of the sugar derived from the food and from endogenous sources, 
to defective oxidation of sugar, to abnormal permeability of the renal 
filter (phloridzin diabetes), or to an excessive ingestion of glucose, 
exceeding temporarily the capacity of oxidation and storage (aliment- 
ary glycosuria) . 

Glycosuria may be produced by the administration of the extracts of 
certain glands, notably the adrenal and the thryoid, and by the use of 
drugs, such as phosphorous, arsenic and ether. It may be seen in 
brain injuries, after severe burns and in cirrhosis of the liver. 

It is evident from the enumeration of the conditions under which 
glycosuria occurs, that it may be transient or permanent. When sugar 
is found in the urine, it is well to repeat the test on another day and to 
be certain that the manifestation is not a transitory or accidental 
matter. The need for caution in this matter is well recognized by 
insurance companies. 

Clinically the glycosuria of greatest interest is that associated with 
15 



226 EXAMINATION OF URINE 

diabetes mellitus, where sugar accumulates in the blood to be excreted 
by the kidneys because of deficient oxidation and storage in the body, 
due in many cases to pancreatic disease. In contradistinction to 
transitory glycosuria, the glycosuria of diabetes is accompanied by 
definite symptoms. In diabetes the quantity of urine excreted is 
large, occasionally reaching 20 liters, and the amount of sugar may 
vary from a trace to as much as 10 per cent. The urine is usually 
pale, acid, and of high specific gravity. Acetone and diacetic acid 
may be present in varying amounts and albumin is commonly seen. 

Detection of Other Sugars.— From a clinical standpoint, glucose is 
the only sugar found in the urine sufficiently frequently to make it of 
especial significance. Lactose is occasionally seen in the urine of 
nursing mothers especially when there is an obstruction to the flow of 
milk, or when nursing has been stopped. Lactose reduces Fehling's 
or Benedict's solutions, but does not give the fermentation test. 
Therefore when a reducing substance is found in the urine of nursing 
mothers, lactose should be thought of and should be carefully excluded 
before making a diagnosis of diabetes mellitus. In carrying out this 
test, it is important to use a pure culture of yeast to prevent bacteria 
ordinarily present in the commercial compressed variety from breaking 
down the lactose into a fermentable sugar. If a pure culture be not 
available, the fermentation test may be performed with the ordinary 
compressed yeast provided that the test be interrupted at the end of a 
few hours, when glucose should give some evidence of gas formation 
while lactose should not. In the amount in which it is present in the 
urine, it is not at all likely to give the phenylhydrazin test. 

The determination of maltose has no practical clinical application. 
It may be demonstrated by determining the nitrogen content of the 
osazone formed, or by the discrepancy between the quantitative 
estimation as performed by the polariscope and by the reduction tests, 
the dextrorotary power being much greater than that of glucose. 

MICROSCOPIC EXAMINATION. 

The microscopic examination of the urine is of great importance 
and should not be omitted when albumin is absent. It should form 
part of the routine examination, and time spent in this, direction will 
benefit the practitioner much more than that spent in most quantitative 
determinations. It is well recognized that a characteristic of the urine 
in chronic interstitial nephritis is the absence of albumin in a urine of 
low specific gravity with the presence of hyaline casts. Then too, the 
presence of pus cells, which may not be present in sufficient quantity 
to give the albumin tests, may give an invaluable clue to infection in the 
urinary tract. 

The student should take pains to examine as many normal specimens 
as possible, so that he may become thoroughly familiar with normal 
findings, and may come to know from experience the number of 



MICROSCOPIC EXAMINATION 227 

leukocytes and epithelial cells which are to be expected in male and 
female urine. The latter is of particular importance, since the normal 
findings are quite different in the two sexes when voided specimens are 
examined, though catheterized specimens are practically the same under 
normal conditions. 

The urine should be centrifugalized in a centrifuge tube with a conical 
tip (Fig. 61, d). If a centrifuge be not accessible, it may be allowed 
to stand for several hours, in a vessel with a conical bottom (Fig. 61, c). 

In order to remove sediments press the pipette firmly at one end with 
dry forefinger and lower the other end to the bottom of the containing 
vessel. Rotating the pipette between thumb and forefinger will allow 
a little of the sediment to enter the pipette. Withdraw the pipette 
and allow the contents to escape on to a glass slide. This is now ready 
for examination under the microscope. 

Darken the field by lowering the Abbe condenser and by almost 
completely closing the iris diaphragm just beneath the stage. First, 
examine the slide without a cover-slip, using the low power, to hunt 
for casts and crystals. Then put on a cover-slip and with high power 
examine for leukocytes, red blood-cells, epithelium and bacteria. A 
well-darkened field is absolutely essential if hyaline casts are to be 
found. 

The constituents to be examined for may be divided as follows : 

1. Organized elements, including casts, cylindroids, epithelial cells, 
leukocytes or "pus-cells," red blood cells or shadows of red cells, 
spermatozoa, mucous cylindroids, bacteria, yeasts, animal parasites. 

2. Unorganized elements, including uric acid crystals, sodium urate 
(amorphous and crystalline), ammonium urate, hippuric acid, ammon- 
ium magnesium phosphate (triple phosphate) crystals, amorphous 
phosphates, calcium phosphate crystals, calcium oxalate crystals, 
calcium carbonate crystals, cystine, leucine, and tyrosine. 

Organized Elements.— Casts.— Casts are formed in the urinary 
tubules, and in reality constitute an impression or casting of the 
tubule, which serves as a mould (Plate V). The substance of a cast 
is probably colloidal, whose exact source, whether a transudate from 
the blood or material from degenerated renal cells, is not settled. 
Casts, especially hyaline casts, do not possess great refractility and 
will be readily overlooked unless the field be well darkened. No 
significance should be attached to the absence of casts in alkaline urine, 
since they dissolve readily in an alkaline medium. The cast is a pale 
cylinder with slightly rounded ends. The relation between diameter 
and length is quite variable. Various types of casts are noted, the 
classification being based on morphology. When the cast is filled with 
granules it is referred to as a finely or coarsely granular cast according 
to the size of the granules. 

There are also blood casts, pus (or leukocyte) casts, epithelial casts, 
fatty casts, depending upon the morphological element which has been 
incorporated in the hyaline matrix of the cast itself. These are of 



228 EXAMINATION OF URINE 

significance since they indicate that the particular morphological 
element has been incorporated in the kidney tubule itself and, therefore, 
has been "shed," as it were, in this place and not lower in the urinary 
tract. Waxy casts are light yellow, have sharply defined outline, and 
are much more refractile. They show a tendency to transverse 
splitting. Fibrin casts are highly refractile and yellowish brown in 
color, occasionally showing ragged fractures. Cells may be incor- 
porated within their substance. They are not actually composed of 
fibrin. They are seen in acute renal conditions and should not be 
confused with waxy casts, which are of much more serious import. 
Fatty casts contain epithelium, which has undergone fatty degeneration. 
Often degeneration is so far advanced that cellular details cannot be 
made out. The droplets stain red with Sudan III or black with osmic 
acid. 

Mucous Cylindroids.— These are long tapering bodies which are thinner 
than either casts or cylindroids. 

Significance of Casts in the Urine.— Hyaline casts are found in various 
forms of nephritis. Their presence is not to be interpreted as absolute 
evidence of the existence of nephritis, since they may be found where 
there is only a temporary irritation of the kidneys (as after unusual 
exercise, during severe infection, or ether anesthesia); indeed, when- 
ever the kidney undergoes marked circulatory change or toxic disturb- 
ance. 

Granular casts, however, usually have more serious import, especially 
if found repeatedly, and may be interpreted as indicative of nephritis. 
It has been claimed that the coarser the granules, the more severe is 
the nephritis. 

Fatty and waxy casts are both found in extremely advanced forms 
of nephritis. Waxy casts are not necessarily a sign of waxy kidneys, 
indeed, the amyloid nature of this form of casts cannot be demonstrated 
in all cases. The occurrence of this form of cast is a sign of grave 
prognostic import. Blood, epithelial, and pus casts point to the fact 
that the incorporated cells have been shed in the kidney itself and their 
presence must be interpreted accordingly. Epithelial casts indicate 
a destruction of renal epithelium and are seen in advanced nephritis. 
Pvs and leukocyte casts point to an infectious process in the kidney 
substance, frequently a pyelonephritis. 

Cylindroids.— Cylindroids resemble casts in their chemical nature and 
general structure except that they taper at one or both ends. It is 
possible for this tail-like end to be broken off so that the remaining 
portion of the cylindroid will present the exact appearance of 
hyaline cast. Differentiation cannot be made in such cases, except 
indirectly, by careful search of the sediment and finding other similar 
elements. The significance of cylindroids is not settled, but it is 
rather generally accepted that their presence does not point to renal 
damage (Plate V). 



MICROSCOPIC EXAMINATION 229 

Mucus Threads.— These are classed by many writers with the cylin- 
droids. They are even less refractile, and are long, thin, tapering- 
threads of ribbon-like rather than cylindrical structure. Chemically, 
they differ from cylindroids, being of mucoid character. They com- 
pose the nubecula. No diagnostic significance is attached to them. 

Leukocytes.— Leukocytes should be readily recognized, particularly 
when the high power of the microscope is employed, bearing in mind 
the presence of a nucleus and the granular appearance of the cyto- 
plasm. The leukocytes are well preserved in acid urine, but tend to 
dissolve in alkaline urine. The addition of a drop of dilute (5 per 
cent.) acetic acid accentuates the nuclei of the leukocytes, making 
them appear darker, while it hemolyzes the red blood cells. Occa- 
sional leukocytes are seen in normal urine, the number being somewhat 
greater in specimens from women. 

An increased number of leukocytes (pyuria) occurs in infection of 
the genito-urinary tract. There is nothing about the number or 
appearance which may be taken as characteristic either of the type of 
infection or the locality involved. 

Red Blood Cells.— Generally speaking, red blood cells are never 
seen in normal urine. When red cells are present in the urine, the 
condition is spoken of as hematuria, and is to be distinguished from 
hemoglobinuria, in which only free hemoglobin is present. 

Red blood cells may be distinguished from white cells with the higher 
power of the microscope, and with the aid of acetic acid, as has been 
mentioned previously. The corpuscles may appear as mere " shadows," 
due to laking of the corpuscles. 

Red blood cells are seen in large numbers, of course, at the time of 
the menstrual period. Pathological conditions which may lead to 
their appearance are the presence of calculi, injuries to the genito- 
urinary tract or the kidneys, inflammatory processes, notably tuber- 
culosis, malignant disease of the kidneys or genito-urinary tract, of 
which papilloma of the bladder serves as an example, acute infectious 
nephritis, acute toxic nephritis, notably that due to mercury, where 
erythrocytes may be seen, even after the therapeutic use of the drug. 
Red cells may be seen in the chronic forms of nephritis, especially with 
acute exacerbations, in chronic congestions of the kidney, such as that 
occurring during decompensation of chronic heart disease, in the 
various form of purpura, occasionally in hemophilia when the hemor- 
rhage is in the genito-urinary tract, and in infection with filaria or 
distoma hamatobium. There is also a so-called "functional" or 
idiopathic hematuria. 

Epithelial Cells.— These are normally seen in small numbers. In 
female urines which have been voided, the normal number is large. 
These are pavement epithelial cells from the introitus. Epithelial 
cells (Plate V) should be divided into three classes : 

(a) Large flat cells, the form most frequently seen, may come from 
ureters, bladder, or vagina. 



230 EXAMINATION OF URINE 

(b) Round cells, rather larger than leukocytes with large, clearly 
denned nuclei. These come from the uriniferous tubules and from 
deeper layers of the mucous membrane of other portions of the urinary 
tract. 

(c) Caudate cells, with distinctly tail-like prolongation of the cell 
body, usually coming from the renal pelvis or the neck of the bladder. 

It should be remembered that the cell forms are not definitely 
characteristic of any one locality in the genito-urinary tract. To 
attempt to localize the exact site of inflammatory process in the genito- 
urinary tract by the identification of a certain type of epithelium is a 
mistake which is not made nowadays by well-informed clinicians. 
The identification of a predominating type of epithelium when taken 
in conjunction with other findings may be of some assistance. 

Yeast Cells.— These are occasionally seen, especially in urines which 
have been kept some time in poorly cleaned receptacles. They are 
not infrequently confused with red blood cells by the inexperienced. 
They should not cause confusion, since the yeast cells are irregular 
in size and frequently show budding. 

Bacteria.— Before drawing conclusions from urinary bacteria, one 
should satisfy himself that the specimen had been voided recently 
into a clean container, and that the organisms are not due to con- 
tamination. 

Unorganized Elements.— There is no doubt that the subject ol 
urinary crystals and amorphous deposits has received vastly more 
attention in the past than has been justified by the clinical knowledge 
derived from the study. Certainly the clinical significance of the 
various forms of crystals is not great. Their identification, however, 
completes the description. Triple and amorphous phosphates are the 
rule in alkaline urines and their presence always suggests the possibility 
of decomposition. Calcium oxalate crystals are observed after the 
ingestion of tomatoes, asparagus, rhubarb, oranges, etc., and are also 
seen in diabetes mellitus, etc. It is stated that calcium oxalate and 
uric acid crystals are found in cases with a tendency toward the 
formation of urinary calculi. Uric acid crystals are seen in a highly 
concentrated, acid urine. Leucine and tyrosine are found in acute 
yellow atrophy of the liver, in acute phosphorus poisoning, in cirrhosis 
of the liver, in cases of severe typhoid. 

Amorphous Deposits.— The gross appearance of the amorphous 
deposits has been described previously. A pink deposit is usually 
composed of urates. It dissolves when the slide is heated gently. 
Urates may be white also. Amorphous phosphates are white in color 
and dissolve readily with the addition of dilute acetic acid. The 
microscopical appearance of an amorphous deposit is the same whether 
it is due to urates or phosphates, the slide looking as though it were 
covered with fine, moderately refractile granules. 

Sediments in Acid Urines (Plate V).— The amorphous sediments 

consist of urates of potassium and sodium. Their appearance has 



PLATE V 




* 


® # 


^m 5 - ? 




<s> 








'J* 




€> 


** ^ 




Urinary Sediments. 

1. Deposit in alkaline or neutral urine: a, ammonio-magnesium phosphate; 6, 
"neutral" calcium phosphate; c, plates of "neutral" calcium phosphate; d, acid ammo- 
nium urate. 

2. Deposit in acid urine: e, calcium oxalate ("dumb-bell" and "envelope" forms); 
/, acid ammonium urate; g, uric acid. 

3. h, vaginal epithelium; i, epithelium from ureter; j, renal epithelium; k, epithelium 
from renal pelvis; I, spermatozoa. 

4. m, hyaline casts; n, cylindroids; o, mucus threads; p, granular casts; q, waxy casts; 
r, fatty cast; s, leukocyte and red blood cell cast. 



MICROSCOPIC EXAMINATION 231 

been described. They dissolve readily with heat or upon the addition 
of acetic acid. 

Uric Acid.— When uric acid separates from the urine, the crystals 
are colored brown, though pure uric acid gives colorless crystals. It 
crystallizes into a number of forms, wedges, whetstones, rhombic 
prisms, irregular rectangular or hexagonal plates, dumb-bells, etc. 
Uric acid crystallizes from an acid urine, particularly when cold. 

Calcium Oxalate.— Calcium oxalate appears as small dumb-bells, or 
more frequently as a refractile, small octahedra with a cross connecting 
the corners. In appearance, the octahedra resemble the back of 
square envelopes. The crystals are precipitated in acid urine, but 
may be seen in decomposed alkaline urine because of their insolubility 
in alkalies. 

Cystine.— Cystine is only rarely found in the urine. It is found in 
acid urines in the form of highly refractile, colorless, six-sided plates. 
Leucine and Tyrosine.— Leucine and tyrosine always occur together 
in the urine, and are found in acid urine in acute yellow atrophy of the 
liver, acute phosphorus poisoning, etc. Leucine crystallizes as small, 
highly refractile, yellowish, spheroids which show both radial and 
concentric striation, while tyrosine forms bundles of fine needles. 

Bilirubin or Hematoidin. — Both substances appear in the form of 
yellow-red needles or rhomboids, soluble in chloroform and potassium 
hydrate. 

Hippuric A cid.— Crystals of hippuric acid are found in the urine 
rather infrequently and their presence is due to the nature of the diet, 
since they are especially apt to be seen after the use of certain fruits 
or the administration of benzoic acid. They appear as colorless needles 
or prisms, pigmented as are the crystals of uric acid as they are found in 
the urine. They are soluble in alcohol and ether. 

Sediments in Neutral or Amphoteric Urine.— In addition to those 
which have been enumerated above as occurring in acid urine, neutral 
calcium phosphate may be found, and rarely, ammonium magnesium 
phosphate. 

Neutral Calcium Phosphate. — Neutral calcium phosphate may appear 
in the form of slender pyramids united at the apices to form rosettes. 
They are soluble in acetic acid and 20 per cent, ammonium carbonate. 
Neutral calcium phosphate also is seen as a scum floating on the top 
of the urine or adhering to the outside of the pipette. Microscopically, 
it appears as large plates or sheets, with very irregular edges. 

Sediments in Alkaline Urine (Plate V).— The amorphous phosphates 
have been described previously. In addition, we may have crystals 
of calcium carbonate, ammonium urate, and of ammonium-magnesium 
phosphate. 

Ammonium-magnesium Phosphate (Triple Phosphates) .—Ammonium- 
magnesium phosphate crystallizes in the form of long prisms (the 
so-called "coffin-lids"), or in long feathery crystals. The latter are 
usually grouped together, four being united at their bases to form a 
letter H. They are readily soluble in acetic acid. 



232 EXAMINATION OF URINE 

Calcium Carbonate.— Calcium carbonate may form amorphous 
masses or may crystallize as spheres or dumb-bells. They dissolve 
readily upon the addition of acetic acid with the evolution of carbon 
dioxide gas. 

Ammonium Urate.— Ammonium urate is ordinarily present in the 
form of dark yellow or brown spheres, often covered with spines. 
They are readily soluble in hydrochloric acid. 

Identification of the Inorganic Sediments.— Chemical means may be of 
assistance in the identification of the sediments. The following table 
given by Sahli is useful in identifying the principal varieties. 
I. Readily soluble upon heating: Urates. 

II. Insoluble or soluble only with difficulty upon heating. 

A. Soluble in acetic acid. 

1. Phosphates (no effervescence). 

2. Calcium carbonate (with evolution of gas). 

3. Ammonium urate (with microscopic precipitate of 

uric acid). 

B. Insoluble in acetic acid. 

1. Soluble in hydrochloric acid; the last three also 

soluble in ammonia, 
(a) Calcium oxalate. 
(6) Leucin, tyrosine, xanthine, cystine. 

2. Insoluble in hydrochloric acid. 

(a) Uric acid. 
(6) Calcium sulphate. 
Another helpful classification is that of Wood, who divides the sedi- 
ments according to the reaction of the urine in which they occur. 
I. Sediments in acid urine: 

A. Amorphous urates (quadrates of sodium and potassium). 

B. Uric acid. 

C. Calcium oxalate. 

D. Cystine. 

E. Leucine. 

F. Tyrosine. 

G. Bilirubin or hematoidin. 
H. Hippuric acid. 

II. Sediments in neutral or amphoteric urine. 

In addition to those above, neutral calcium phosphate may be found 
and ammonium-magnesium phosphate rarely. 

III. Sediments in alkaline urine. 

A. Amorphous phosphates (tricalcium and trimagnesium 

phosphates) . 

B. Calcium carbonate. 

C. Ammonium urate (spheres or "thorn apples"). 

D. Triple phosphates or ammonium-magnesium phosphate. 



SPECIAL QUALITATIVE TESTS 233 



SPECIAL QUALITATIVE TESTS. 



A number of qualitative tests maybe performed on special occasions, 
either when the clinical findings or when the laboratory data obtained 
on the routine examination point to their desirability. 

Tests for Bile Pigments.— When the urine is highly colored, when the 
foam shows yellow color, when jaundice is present, or when clinical 
indications suggest an obstruction of the biliary tract or inflammatory 
conditions of the gall-bladder or biliary passages, tests for bile should 
be made. 

Foam Test.— Shake the urine in a bottle. A brownish-yellow foam 
indicates the presence of bile. This test should not be accepted as 
final, but should be confirmed by one of those given below. 

Gmelin's Test.— Under the 5 cc. of urine in a test-tube, run from a 
pipette 5 cc. of concentrated nitric acid, carefully and slowly, as in 
Heller's test for albumin. If bile be present, there will appear con- 
centric rings of various colors. 

Trousseau's Test.— With a pipette run 5 cc. urine under 5 cc. of very 
dilute tincture of iodine (1 part of tincture of iodine plus 39 parts of 
95 per cent alcohol) . Bile pigments will give a dark-green ring at 
the junction of the liquids. 

Significance of Bile Pigments.— Bile pigments may gain access to the 
general circulation and be excreted by the urine whenever there is an 
obstruction of the free outflow of bile from the liver into the intestinal 
tract. This obstruction may be in the common duct, in the smaller 
biliary passages, or in any of the intermediary passages. Mechanical 
causes are stones in the biliary passages, pressure from without by new 
growths such as a tumor of the pancreas, a tuberculous lymph gland, 
or adhesions. Jaundice is a conspicuous sign in the acute catarrhal 
jaundice accompanied by gastro-intestinal disturbances, and is seen in 
cirrhosis of the liver, acute yellow atrophy, anemia of hemolytic 
jaundice, and in cholangitis. 

Determination of Bile Acids.— The determination of bile acids requires 
the presence of at least 0.5 per cent. This impairs the value of the 
procedure, since this amount is often not present. The detection 
of bile pigments is therefore much the method of choice on account of 
the possibility of detecting readily much smaller traces. The detection 
of both pigments and acids is a needless refinement since pigments and 
acids are usually present under the same conditions. 

There is no test for bile acids which is both simple and reliable. 
Hay's tests depended upon an alteration in the surface tension. Flour 
of sulphur is dusted over the surface of the urine, which is placed in a 
beaker or open vessel. It was assumed that the sulphur would sink 
because of the reduction in surface tension when bile acids are present 
and would float on the surface when absent. 

Tests for Acetone and Diacetic Acid.— Examination should be made 
for these bodies whenever sugar is found on the routine examination, 



234 



EXAMINATION OF URINE 



when the clinical history indicates diabetes mellitus, or when the 
patient is in coma. 

Tests for Acetone.— In performing any of the tests for acetone except 
Rothera's, the urine should be distilled, using either a retort or a flask 
fitted with a cork through which is passed a long piece of glass tubing 
(Fig. 64). The urine is acidulated with 5 per cent, acetic acid and 
distillation is allowed to proceed until about one-third of the original 
volume has been collected. To the distillate add 5 drops of 10 per cent. 
HC1 and redistill. 

Rotheras Test.— Preference is given to this method. To 5 or 10 cc. 
of urine add about 1 gm. of ammonium sulphate and 2 or 3 drops of a 
freshly prepared 5 per cent, solution of sodium nitroprusside. Stratify 




Fig. 64. — Distillation of urine for acetone tests. 



strong ammonium hydroxide upon the mixture. When acetone is 
present, a characteristic permanganate color develops slowly. The 
delicacy of the test is 1 to 20,000. 

Gunning's Iodoform Test.— To 5 cc. of distillate add a few drops of 
Lugol's solution and enough ammonia to form a black precipitate. 
When the black precipitate appears, add a few more drops. Allow the 
tube to stand until a yellowish sediment appears, and examine this 
microscopically for iodoform crystals. These are six-sided plates and 
six-pointed stars. 

LegaVs Test.— To 5 cc. of distillate, add a few drops of a freshly 
prepared concentrated aqueous solution of sodium nitroprusside. 
Render the mixture alkaline with 10 per cent, potassium hydroxide. 



SPECIAL QUALITATIVE TESTS 235 

Normally a red color appears. This is due to creatinine. Add an 
excess of glacial acetic acid and if acetone be present, the color will be 
intensified to almost a purple. If acetone be absent, a yellow color is 
seen. 

Diacetic Acid. — Gerhardt's Test.— Put 5 cc. of urine in a test-tube and 
add 5 per cent, aqueous solution of ferric chloride, drop by drop, as 
long as precipitation occurs. If a heavy precipitate of phosphates 
occur, filter and to the fitrate add a few drops of the ferric chloride 
solution. A Bordeaux red color is produced by diacetic acid. 

The reaction is a general one for ketone bodies, since a reaction is 
given by acetone as well as diacetic acid. 




Fig. 65. — Iodoform crystals. 

Test for /3-Oxybutyric Acid.— Hart's Test— Twenty cc. of urine are 
placed in an evaporating dish and an equal quantity of water is added. 
The mixture is boiled down to about 10 cc. to free it of the acetone and 
diacetic acid. Water is added to bring the volume to 20 cc. and the 
mixture is divided equally between two tubes. One cc. of hydrogen 
peroxide is added to one of the tubes and to each tube is added 0.5 cc. 
of glacial acetic acid, with a small crystal of sodium nitroprusside. 
After mixing, 2 cc. of concentrated ammonia is layered over the mixture 
in each tube and the tubes are allowed to stand. If /3-oxybutyric acid 
be present, the contact ring should show a purplish red color in the 
tube to which hydrogen peroxide had been added, while it should be 
colorless in the control tube to which no peroxide has been added. 

Significance of the Acetone Bodies. — The foregoing substances, beta- 
hydro-oxybutyric acid, diacetic acid and acetone, are closely related 
chemically and the clinical significance of their presence in the urine 
will be considered at the same time. Beta-hydro-oxybutyric acid is 



236 EXAMINATION OF URINE 

the mother-substance, oxidation yields diacetic acid, and oxidation of 
this in turn gives acetone, though diacetic acid is considered the mother- 
substance by some. The chief source of the bodies is from the fats, 
which are improperly metabolized. Fat is less readily oxidized than 
carbohydrates and requires "the fire of burning sugar to consume it. 
If the carbohydrate fires do not burn briskly enough, the fat is incom- 
pletely consumed; it smokes, as it were, and the smoke is represented 
in metabolism by the ketones and derived acids." (MacLeod.) Lack 
of combustion of the carbohydrates may be brought about by deficient 
intake, as in starvation, or by faulty combusion, as in diabetes. The 
accumulation of these acetone substances in the body leads to a type 
of acidosis which is seen in diabetes, cholera, in starvation and in other 
conditions. Obese individuals not infrequently show acetone bodies 
in the urine during preoperative and postoperative starvation. 

In milder cases, only small amounts of acetone may be excreted, but 
in more severe cases diacetic acid and (8-hydro-oxybutyric acid also 
are found in their urine. The amount of these substances present 
serves as a guide to the degree of acidosis. The exact quantitative 
determination is time-consuming, and the amount may be arrived at 
indirectly by the determination of the ammonia, since the diacetic 
acid and /3-oxybutyric acid in the organism combine with ammonia 
and are excreted as ammonia compounds. Magnus Levy states that 
the normal daily excretion of ammonia is 0.7 gm. and that if the 
amount is raised to 2 gm. it is equivalent to about 6 gm. of oxybutyric 
acid, while 5 gm. is equivalent to 20 gm. of oxybutyric acid. 

The continued finding in the urine of all three acetone bodies with an 
increased amount of ammonia and the persistence of these bodies in 
spite of withholding carbohydrates, is rather generally accepted as 
basis for a poor prognosis. These findings may have little relation to 
the quantity of sugar excreted. In mild cases, the amount of sugar 
may be small, and may disappear upon limitation of the carbohydrate 
intake. With this limitation of carbohydrates, the amount of acetone 
may increase but diminishes as the organism becomes accustomed to 
burning fat. 

Indicant.— Obermayer's Test.— In a test-tube place about 10 cc. of 
clear urine and 10 cc. Obermayer's reagent (made by adding 2 gm. 
of ferric chloride to 1000 cc. hydrochloric acid) and add 5 cc. of chloro- 
form. Shake well and cork the tube. If indican be present, the 
chloroform will take the color of indigo blue. Normal urine shows 
only a faint blue color. The depth of the coloration serves as an 
approximate measure of the amount of indican present. If desired, 
the urine may be mixed with the hydrochloric acid and two or three 
drops of 5 per cent, ferric chloride may be added instead of using 
Obermayer's solution. Indican is derived largely from the putrefac- 
tion of protein which occurs in the intestinal tract, protein cleavage 
resulting in the formation of indol and skatol. Indol is absorbed in 
the circulation to indoxyl, which is conjugated in the liver with sul- 



SPECIAL QUALITATIVE TESTS 237 

phuric acid to form indoxyl-potassium-sulphate, or indican. Increased 
quantities are found in the urine in conditions which permit intestinal 
putrefaction, such as intestinal obstruction, ileus, and peritonitis. 
It is only occasionally found in cases of simple constipation. When 
the large bowel is occluded, indican does not appear unless there be also 
stasis of the ileum. Decomposition of protein in other parts of the 
body may give rise to an increase in the amount of urinary indican, 
such as gangrene of the lung, empyema, etc. 

While indican may be determined quantitatively by Ellinger's 
method, the technic is not such as would recommend it for clinical work. 
The clinical value of the indican test, performed qualitatively, is rather 
limited and is seen chiefly in connection with mild gastro-intestinal 
disturbances of the functional type, marked by symptoms of headache 
and lassitude. 

Blood.— Blood may occur in the urine in the unchanged form, when 
the corpuscles may be identified microscopically, the condition being 
referred to as hematuria; or it may be excreted as a solution of 
hemoglobin or one of its derivatives, this condition being referred to 
as hemoglobinuria. The detection of red blood corpuscles has been 
discussed previously. Hemoglobin is easily identified by the spec- 
troscopic method. Since hemoglobin is a protein, urine containing it 
would give the usual tests for protein bodies. 

Benzidine Reaction.— About 2 cc. of urine are placed in a test-tube 
and are acidified with glacial acetic acid. If the blood be unchanged, 
this serves to lake the corpuscles and to convert the hemoglobin to 
acid hematin. About 5 cc. of an alcoholic solution of benzidine are 
added (prepared by making a saturated solution in 95 per cent, alcohol 
heated over a water-bath) . The contents of the tube are thoroughly 
mixed and 5 cc. of hydrogen peroxide solution are added. A Prussian 
blue color indicates the presence of blood pigment. Care should be 
taken that the solution of hydrogen peroxide is a potent one. 

The test has been simplified by the use of compressed tablets con- 
taining benzidine and sodium perborate according to the method 
of Roberts. A tablet is soaked with the urine or with a watery emul- 
sion of the feces to be examined, and upon it is then placed a drop of 
glacial acetic acid. The oxygen is furnished by the sodium perborate 
instead of by hydrogen peroxide. This modification has the advantage 
of ready availability and simplicity, but is not quite as delicate as the 
method previously described. 1 

Spectroscopic Test. — A small pocket spectroscope is required (Fig. 66). 
The urine should be filtered and placed in a test-tube and a layer of 
distilled water run over it with care to avoid shaking, so that gradual 
mixing may give a column of fluid with varying dilution of hemoglobin 
from the bottom to the top of the tube. The tube is placed in position 
in the holder of the spectroscope and the absorption bands are located, 

1 These tablets are prepared by E. R. Squibb and Sons. 



238 



EXAMINATION OF URINE 



using direct daylight. The bands may be those of oxyhemoglobin, 
of reduced hemoglobin, of methemoglobin, or rarely, of hematin. 

Oxyhemoglobin shows two bands between D and E; reduced hemo- 
globin, one band between D and E; methemoglobin, if the solution be 
acid, by an additional dark band between C and D (Plate VI). 

If the worker be not entirely familiar with the spectra in question, 
it would be advisable to prepare control tubes with known solutions 
for the sake of comparison. Oxyhemoglobin would be prepared by 
making a dilute solution of blood in distilled water. Reduced hemo- 
globin may be prepared by taking blood which has been diluted to 
show the two absorption bands of oxyhemoglobin and by adding to it a 
few drops of Stoke's reagent (a solution made with ferrous sulphate, 
2 gm.; tartaric acid, 3 gm.; distilled water, q. s., to 100 cc. When 
needed for use, ammonium hydroxide is added to a small quantity 




Spectroscope. 



drop by drop until the precipitate which forms at first entirely dis- 
solves). Methemoglobin may be prepared by diluting a drop or two 
of blood with distilled water. To the dilution is added a few drops of 
freshly prepared 10 per cent, solution of potassium ferricyanide, and a 
few drops of acetic acid to acidulate. 

Significance of Hemoglobinuria.— The significance of hematuria has 
been discussed. Hemoglobinuria implies the presence of free hemo- 
globin in the circulating blood. This may occur after transfusions of 
blood, in severe form of malaria (so-called "black-water fever"), as 
a result of hemolytic poisons, such as snake venom, nitrobenzol, chloro- 
form, pyrogallic acid, etc., and in paroxysmal hemoglobinuria. 

llematoporphyrin.— Hematoporphyrin is an iron-free derivative of 
hemoglobin normally found in minute traces. The amount is increased 
in sulphonal, trional, and tetronal poisoning. Spectroscopic examina- 
tion is employed for its identification. 



PLATE VI 



C D 


E b F 


1 












J 










2 


I 
i 




,!;-,. | 






















3 


, 




i i ! 


WSSm 






i 






4 


m\ j$ 


(■m™ I 
















5 
























6 




y ■ i 




'-.,:, .-. - 


i 














7 




I ! 




















8 








|'-...^:' ( 


1 












>j 


9 






I ' 



Absorption Bands of Hemoglobin and Some of its Derivatives. 

Absorption bands of (1) oxyhemoglobin; (2) reduced, hemoglobin; (3) neutral met- 
hemoglobin;(4) alkaline methemoglobin; (S) carbon monoxide hemoglobin; (6) alkaline 
hematin; (7) acid hematoporphyrin; (8) urobilin or hydrobilirubin in acid solution; (9 
urobilin or hydrobilirubin in alkaline solution after adding zinc chloride. 



SPECIAL QUALITATIVE TESTS 239 

Procedure.— About 20 cc. of 10 per cent, sodium hydrate are added to 
100 cc. of urine. The resulting mixture is filtered through filter paper, 
the filtrate discarded, and the precipitate washed with distilled 
water and partially dried by blotting it with filter paper. It is then 
scraped into a test-tube with a spatula and is dissolved by shaking 
with a mixture containing about 1 cc. of hydrochloric acid and 9 cc. of 
95 per cent, alcohol. The solution should be filtered and examined 
with the direct vision spectroscope for the absorption bands of acid 
hematoporphyrin, one a narrow band slightly to the left of the D line 
and the other a much broader band between the D and E lines. 

A control may be prepared by adding two drops of blood to 5 cc. of 
concentrated sulphuric acid. 

Urobilin.— Ten cc. of urine are alkalinized with ammonia and 25 
drops of 10 per cent, solution of zinc chloride are added. The mixture 
is allowed to stand a few moments when it is filtered to free it from 
phosphates. To the filtrate are added a few more drops of the zinc 
chloride solution. The tube should be held against a dark back- 
ground and examined with a ray of strong light. An electric pocket 
flash light or the light from a head mirror may be utilized. When 
urobilin is present a distinct green fluorescence is seen. 

The urine may be examined directly with the spectroscope. The 
absorption bands may be intensified somewhat by adding a few drops 
of tincture of iodine. In an acid solution the band is in the green 
portion, between the lines b and F. If desired, the zinc chloride test 
may be confirmed spectroscopically. The band will be broader and 
will be moved over somewhat to the left hand side of the spectrum, so 
that it will overlap the b line. 

Urobilin or its precursor urobilinogen, exists in all urine. Its origin 
is not definitely settled, but the weight of opinion inclines to the follow- 
ing explanation. In the destruction of the erythrocytes, the liberated 
hemoglobin is carried to the liver, where the chromogen group yields 
the bile pigment, bilirubin. This is excreted as urobilinogen and 
urobilin. Both are reabsorbed into the blood and are excreted into the 
urine, which normally contains urobilin or urobilinogen. The excre- 
tion is greatly increased in certain conditions, in the hemolytic anemias, 
in the poisonings and in the infectious diseases characterized by des- 
truction of blood, in cirrhosis of the liver, in acute yellow atrophy of 
the liver, in hemophilia, extra-uterine pregnancy, secondary syphilis, 
etc. 

It is absent from the urine in cases of advanced nephritis where the 
impairment of the kidney prevents its excretion into the urine (Wood), 
and in cases where a complete obstruction of the common bile duct 
prevents the excretion of billirubin into the intestines and the conse- 
quent formation of urobilin (Barker). 

From what has been said, it may be seen that a positive result with 
a qualitative test does not have any great clinical significance since 
urobilin or urobilinogen is found in the normal urine. A quantitative 



240 EXAMINATION OF URINE 

test might furnish information of considerable value serving particu- 
larly as an index of increased blood destruction, but because of its 
technic, it does not commend itself for clinical purposes. Barker feels 
that a negative qualitative test gives important confirmatory evidence 
of the presence of an icterus due to obstruction of the common duct. 

Diazo Reaction.— Ehrlich's reagent should be freshly prepared by 
mixing one part of solution A with 50 parts of solution B. Equal 
quantities of this reagent and urine are placed in a test-tube and 
thoroughly shaken. The test is regarded as positive when the fluid 
assumes a deep cherry-red color, and the foam is distinctly pink. 
Normal urine results only in a brownish yellow color. A test should 
not be regarded as positive unless the foam be pink. 

The formulae for the two solutions are : 

A 

Sulphanilic acid 1 gm. 

Hydrochloric acid (concentrated) 50 " 

Water 1000 cc. 

B 

Sodium nitrite 1 gm. 

Water 200 cc. 

The nature of the substances which give this color reaction with 
the diazo bodies is unknown. The reaction is obtained in from 70 to 
SO per cent, of the cases of typhoid in the first two weeks and in relapses. 
Its value is considerably impaired by the fact that positive reactions 
are also given in pneumonia, acute miliary and pulmonary tuberculosis, 
measles, scarlatina, sepsis, and trichinosis (Barker). Certain drugs 
also give the reaction, notably chrysarobin and naphthalin. Wood 
states that these reactions may be distinguished by the fact that with 
them the color is more permanent in alkaline solutions; that it does not 
fade appreciably upon the addition of strong mineral acids; that the 
foam is more yellow; and that a green precipitate does not appear upon 
standing. 

The clinical value of the diazo reaction is extremely limited, for 
it appears in many diverse conditions. As a diagnostic test for 
typhoid fever, it is, in the writer's opinion, of very slight assistance, 
since it is given in a limited number of positive cases and is found in 
so many conditions which are most likely to give rise to confusion in 
making a differential diagnosis. 

"Bence- Jones Protein."— Heat the urine gently, noting the tempera- 
ture. If "Bence-Jones protein" be present, a turbidity may appear 
at as low a point as 40° C. It is convenient to stand a test-tube filled 
with urine in a beaker of water. A thermometer is held in the water, 
which is heated gradually to 100° C. ^lien the temperature reaches 
60° C, flocculent precipitation forms. Acidify the urine slightly with 
dilute acetic acid, and heat to 100° C. Most of the precipitate will 
disappear but will reappear upon cooling. 



SPECIAL QUALITATIVE TESTS 241 

As a confirmatory test, it is well to precipitate the protein by the 
addition of 25 per cent, nitric acid. This precipitate should clear 
on heating and reappear on cooling. Further, the protein is com- 
pletely precipitated by adding to the urine 2 volumes of 96 per cent, 
alcohol. 

It is generally admitted that the Bence-Jones body is not an albu- 
mose, as it was at first regarded, but is a true protein, elaborated in the 
body under abnormal conditions, yielding on digestion primary and 
secondary albumoses, and characterized by a high aromatic radical 
content. It is found in many, though not all, cases of multiple mye- 
loma, especially when the thoracic skeleton is involved (Simon), and 
occasionally in leukemia. Its occurrence has been reported in isolated 
cases of osteomalacia, carcinomatosis involving the osseous system, and 
one case of gunshot wound of leg. 

Miller and Baetjer report that in a period of eighteen months they 
found seven cases of Bence-Jones proteinuria. One was a case of 
chronic myelogenous leukemia, one a case of true myeloma, and in the 
other cases there were no evidences of either of these diseases. Some 
of the latter were young and apparently healthy individuals showing 
hypertension and cylindruria despite renal function tests which gave 
apparently normal results. They advance the idea that these cases 
may be additional proof that Bence-Jones proteinuria may be an 
inborn error of metabolism. 

They also suggest that possibly many instances of this form of pro- 
teinuria are missed in the urinary examinations as routinely performed 
simply because of the widely accepted idea of its rarity. 

Nucleoprotein. — Place 10 cc. of urine in a beaker, add 3 volumes 
of water to prevent the precipitation of urates and acidify strongly acid 
with acetic acid. Turbidity indicates the presence of nucleoprotein. 
Positive identification is difficult. If albumin be present, it should 
be removed by boiling and filtering the urine before testing it. 

Another method which has been employed is to perform the cold 
nitric acid test. Albumin or globulin will give a compact ring at the 
line of junction between urine and acid, while the precipitate of nucleo- 
protein is usually more diffuse, and will rise to a higher level, about 
5 to 10 mm. above this line. 

The chemistry of the nucleoprotein and nucleoalbumin bodies 
occurring in the urine is decidedly a moot question. A survey of the 
current literature and standard texts show that no two writers agree 
as to the nature of the substance which is precipitated by acetic acid in 
diluted urine in the cold, and that the identification of the bodies has 
not been completed by demonstration of the nuclein and phosphorus 
portions. Whatever may be the chemical structure and relationship 
of the bodies, it must be admitted that their clinical importance is not 
great, the important point being to avoid confusing them with serum 
albumin or globulin, whose presence in the urine is of so much greater 
import. This fact is recognized by certain of the insurance companies, 
16 



242 EXAMINATION OF URINE 

which will accept applicants whose urine shows " nucleoprotein" as 
recognized by the foregoing tests while an albuminous urine would be 
ground for rejection. 

QUANTITATIVE DETERMINATIONS. 

Cautions.— It should be remembered that any quantitative deter- 
mination has little value unless it is made with a twenty-four-hour 
specimen, in order that the total daily output of a given substance 
may be determined. 

Albumin.— Esbach's Method.— An Esbach albuminometer tube is 
necessary (Fig. 63, A). Introduce urine to the mark U, and then 
carefully add Esbach's reagent (10 gm. of picric acid and 20 gm. of 
citric acid dissolved in 1 liter of water) to the mark R. Cork the tube, 
invert gently about 10 times so that mixing may be thorough, and 
place the tube in a test-tube rack, where it should stand for twenty-four 
hours. Then the upper level of the precipitate may be read on the 
engraved scale, which gives the number of grams of protein per liter 
of urine. If the precipitate comes to the mark 2, there are 2 gm. of 
protein per liter, or 0.2 per cent. 

Tsuchiya's Method.— This method is more accurate and may be 
employed in the same way. The formula is: phosphotungstic acid, 
1.5 gm.; concentrated hydrochloric acid, 5 cc; alcohol (95 per cent.), 
95 cc. 

Purdy's Method.— Put 10 cc. of urine in a 15 cc. graduated centri- 
fuge (Fig. 54, g) tube and add 3 cc. of 10 per cent, potassium ferro- 
cyanide and 2 cc. of 50 per cent, acetic acid. Mix solutions thoroughly 
and allow the tube to stand for ten minutes. Centrifugalize for exactly 
three minutes at 1500 revolutions per minute in a centrifuge the 
radius of which is 6f inches to the tip of the tube. The amount of 
precipitate is noted and the result translated to grams of protein per 
liter of urine from the following table : 

Volume of Volume of 

precipitate Dry weight precipitate Dry weight 

in graduated of protein in graduated of protein 

tube, to liter, . tube, to liter, 

cc. gm. cc. gm. 

0.25 0.5 2.75 5.7 

0.50 1.0 3.00 6.3 

0.75 1.6 3.25 6.8 

1.00 2.1 3.50 7.3 

1.25 2.6 3.75 7.8 

1.50 3.1 4.00 8.3 

1.75 3.6 4.25 8.9 

2.00 4.2 4.50 9.4 

2.25 4.7 4.75 9.9 

2.50 5.2 5.00 10.4 

Sugar.— Fermentation Method.— -This method is only approximately 
accurate, but has the advantage of simplicity. An Einhorn sacchar- 
ometer is required (Fig. 63, b). About 15 cc. of urine are placed in a 



QUANTITATIVE DETERMINATIONS 243 

beaker and in it is ground up about one-sixteenth of a cake of com- 
pressed yeast, using a glass rod as a pestle. The resulting mixture is 
placed in a saccharometer tube. Care must be taken that there are no 
air-bubbles at the top of graduated tube, which should be completely 
filled with urine. The tube is placed in a warm room or in the incuba- 
tor, and at the end of twelve hours the percentage of sugar is read at the 
upper level of the fluid in the tube. A control tube should be set up 
with normal urine mixed with the same amount of yeast. 

If the specific gravity is high, pointing to the presence of a large 
quantity of sugar, the urine should be diluted by adding 1, 2, 3, or 4 
times the amount of distilled water, to make a dilution whose specific 
gravity will be about 1.010. The final reading should be multiplied 
by 2, 3, 4, 5, as the case may be. 

Lohnstein Saccharometer.— The instrument consists of a glass bulb 
connected with a U-tube, one arm of which is longer than the other 
and is fitted with two scales on which the percentage of sugar may be 
read, one adjusted for room temperature and the other for incubator 
temperature. The outfit should contain 12 cc. of mercury, which 
serves to separate the urine from the atmosphere. One-half cc. of 
urine is placed in the bulb over clean mercury and the same amount of 
thick emulsion of yeast is added. After the stopper has been greased 
with a mixture of equal parts of vaseline and yellow wax, it is inserted 
so that the apertures in the stopper and the neck of the bulb corre- 
spond, allowing communication between the space inside the bulb and 
the outside air. The apparatus is then tipped so that the level of the 
mercury in the long arm coincides with the zero of the scale, when the 
stopper is turned enough to close the opening. A weight is placed on 
the stopper and the apparatus put in the incubator for six hours, when 
the result may be read at incubator temperature on the scale adjusted 
to 35° C. When an incubator is not available, fermentation may be 
allowed to proceed about twenty-four hours at room temperature. 
The chief disadvantage is the difficulty in cleaning. 

Reduction Methods. — The reduction methods are generally employed. 
A number have been devised, all of which are modifications of Fehling's 
test. Purdy and Pavy's methods have certain points of advantage, 
chief of which is the exceedingly sharp end-point. Purdy 's solution 
keeps for a period of months, much better than any of the copper 
solutions proposed prior to Benedict's. The principal objections to 
it are the liberation of ammonia fumes, which are annoying in a labora- 
tory, and the lack of permanence as compared with Benedict's solution. 
The method of Benedict is almost ideal and has come into general use 
in this country. If the estimation be carried out according to Myers' 
suggestion with the use of a small quantity of the reagent, the per- 
formance of a determination becomes a matter of extreme simplicity. 

Fehling's Quantitative Method.— Equal quantities of Fehling's copper 
sulphate solution and alkaline tartrate solution are measured with a 
volumetric pipette and mixed. The copper sulphate solution must be 



244 EXAMINATION OF URINE 

made with great care, weighing the copper sulphate accurately. The 
urine is diluted by placing 10 cc. in a 100 cc. volumetric flask, adding 
water to bring the volume to 100 cc, and placed in a burette after 
thorough mixing. In a beaker, 10 cc. of Fehling's solution and 40 cc. 
of water are boiled over a flame. The burette is clamped over the 
beaker and when the Fehling's solution has reached the boiling-point, 
diluted urine is run from the burette until cupric oxide has been 
completely precipitated as red cuprous oxide, the end-point being indi- 
cated by disappearance of all blue color. The chief objection to the 
method is the uncertainty of the end-point. At least three deterinina- 
tions should be made, and the average taken of the readings. 

Ten cubic centimeters (10 cc.) of Fehling's solution are reduced by 
0.05 gm. dextrose (or 0.0676 gm. lactose). The fluid used for titration, 
therefore, contained 50 gm. of glucose (or 67.6 gm. lactose). If the 
dilution of urine were made as prescribed (1+9), this formula may be 
employed for ascertaining percentage : 

50/ X= percentage in original sample when X represents the number 
of cc. of diluted urine required to reduce the stated amount of reagent. 

Preparation of Benedict'* Reagent.— 

( Jopper sulphate (crystallized) 18 gm. 

Sodium carbonate (crystallized; one-half the weight of the 

anhydrous salt may be used) 200 " 

Sodium or potassium citrate 200 " 

Potassium thiocyanate 125 " 

Potassium ferrocyanide (2 per cent, solution) 5 cc. 

Distilled water to make a total volume of 1000 " 

With the aid of heat dissolve the carbonate, citrate and thiocyanate 
in enough water to make about 800 cc. of the mixture and filter if neces- 
sary. Dissolve the copper sulphate separately in about 100 cc. of 
water and pour the solution slowly into the other liquid, with constant 
stirring. Add the ferrocyanide solution, cool and dilute to exactly 
1 liter in a volumetric flask. Of the various constituents, the copper 
salt only need be weighed with exactness. Twenty-five cc. of the 
reagent are reduced by 50 gm. of glucoso. 

Procedure. — Ten cc. of urine are diluted in a volumetric flask to 
100 cc. with distilled water, and a burette filled with the resulting 
mixture. Into a porcelain evaporating dish (about 25 to 30 cm. in 
diameter) 25 cc. Benedict's reagent are measured with a pipette, and 
10 to 20 gm. crystallized sodium carbonate are added. One-half this 
amount should be used if the anhydrous salt be employed. A little 
powdered pumice or talc is added, and the mixture heated over a free 
flame until the carbonate has dissolved (Fig. 67). Then the diluted 
urine is quickly run in from the burette until the last trace of blue has 
gone. Throughout the titration the mixture must be kept boiling; 
should it become too concentrated, water may be added. 

Calculation. Twenty-five cc. of Benedict's reagent are reduced 
by 50 mg. glucose (67.6 mg. lactose, 74 mg. maltose, 47.5 mg. invert 



QUANTITATIVE DETERMINATIONS 



245 



sugar). Therefore, the fluid used for titration contained 50 mg. of 
glucose. If the dilution of urine were made as prescribed (1+9), this 
formula may be employed for determining the percentage of glucose: 

50/X = percentage in original sample when X represents the number 
of diluted urine required to reduce the stated amount of 
reagent. 

Corresponding factors may be substituted for 50 with other sugars. 




Fig. 67. 



-Arrangement of apparatus for quantitative determination of glucose by 
Benedict's method. 



Benedict's Method Employing Small Quantities of Reagent.— V '. C. 
Myers (personal communication) has suggested the use of smaller 
quantities of reagents. Five cc. of Benedict's solution are placed 
in a test-tube and about 2 to 3 gm. of anhydrous sodium carbonate 
added. The tube is held over the flame against a white background 
until the contents start to boil when urine is added from a Mohr 



246 



EXAMINATION OF URINE 



pipette of 1 cc. capacity graduated in 0.1 ccs. The reagent is boiled 
gently. Meanwhile the urine is run in slowly until the end-point 
described above had been reached (Fig. 68). If the specific gravity 
be high, or if a preliminary determination show high sugar content, the 
urine should be diluted accordingly so that the amount of dilution 
required for reduction would be not less than 1 cc. At least two deter- 
minations should be made, and the average of the results taken. The 
method is readily performed in the simplest clinical laboratory and is 
extremely economical of reagents. 




-Determination of glucose by Myers's modification of Benedict's method. 



Calculation.— Five cc. of Benedict's solution are reduced by 10 mg. 
of glucose. The amount of urine required to produce reduction, 
therefore, contained 10 mg. of glucose. 

If the specimen were undiluted, 1/X gives the percentage of sugar in 
the undiluted urine when X represents the number of cc. of urine 
required to cause reduction. If the specimen were diluted the same 
formula would be used, X representing the number of cc. of diluted 
urine employed, and the result, of course, would be divided by the 
dilution. 



QUANTITATIVE DETERMINATIONS 



247 



Purely' s Method.— Preparation of Solution.— The solution is prepared 
as follows : 

Pure cupric sulphate 4.752 gm. 

Potassium hydroxid . 23.500 " 

Strong ammonia (U. S. P., sp. gr., 0.9) 350.000 cc. 

Glycerol 38.000 " 

Distilled water to 1000.000 " 



Prepare by dissolving the cupric 
sulphate and glycerin in 200 cc. of 
distilled water with the aid of mod- 
erate heat. In another 200 cc. of 
distilled water dissolve the potas- 
sium hydroxid. Mix the two solu- 
tions. Cool and add the ammonia. 
Finally, with distilled water bring 
the volume of the whole to exactly 
1 liter in a volumetric flask. 

Procedure. — Measure 35 cc. of 
Purdy's solution into an Erlenmeyer 
flask of about 250 cc. capacity, and 
add about 100 cc. of distilled water. 
Charge a 25 cc. burette, graduated 
in 0.1 ccs. with the urine, allowing it 
to run out until all the air in the 
terminal and rubber connections 
has been expelled. The Erlenmeyer 
flask should be provided with a 
tight-fitting rubber stopper, perfor- 
ated with two holes (Fig. 69). The 
glass terminal of the burette is 
introduced through one hole, and 
through the other is passed a piece 
of glass tubing about 8 inches long, 
bent at right angles at a point a 
little above its emergence from the 
rubber stopper. This provides a 
vent to carry out the ammonia fumes. 
Mount the burette with attached 
Erlenmeyer flask and fittings on 
the burette stand, supporting the 
flask suitably with the iron ring, 
protecting the bottom with wire 
gauze. With a Bunsen flame, 
bring the contents of the flask to 

the boiling-point, and then slowly discharge urine from the burette 
until the blue color begins to fade; "then still more slowly, three to 
five seconds elapsing after each drop, until the blue color completely 




Fig. 69. — Arrangement of apparatus 
for determination of glucose by Purdy's 
method. 



248 



EX AMI X ATI OX OF URINE 



disappears and leaves the test-solution transparent and colorless." 

This marks the end-point. At least two determinations should be 

made, taking the average of the readings. 

Calculation.— The amount of urine required to reduce 35 cc. of the 

solution contains exactly 0.02 gm. of sugar. The percentage can be 

determined with this formula: 

2 

Y = percentage of sugar, when X represents the number of cc. of 

urine required to effect reduction. 

Example : 2 cc. of urine are required to reach the end-point . 2 -J- 2 = 1 
per cent. 

Precautions.— Should the amount of sugar exceed 3 to 4 per cent., it 
is well to repeat the manipulations with urine diluted with 2 or 3 parts 
of water. In this event the calculation should be corrected accord- 
ingly, dividing the result 3 or 4, as the case may be, before employing 
the formula given above. 




3^~ 



-Polariscope. A, ocular; B, graduated dial; C, lover; D, cap; E, glass i 
F, tube for urine ; G, lamp. 



Quantitative Determination of Glucose by Polariscope. Urine con- 
taining albumin should be acidified with acetic acid, boiled to secure 
complete coagulation and filtered. It should be cleared by adding a 
few crystals of lead acetate and filtering, passing through the same 
filter two or three times if necessary to clarify completely. Before 
using the instrument the position of the zero mark should be adjusted. 
After the polariscope tube has been thoroughly cleaned with distilled 
water and dried perfectly it may be filled with cleared and filtered urine 



QUANTITATIVE DETERMINATIONS 249 

so that the meniscus is convex. The glass disk (Fig. 70) which serves 
as a cover for the end is slid on to displace any excess of fluid, leaving 
the tube full of urine and free from air-bubbles. The cap is screwed 
on and the tube placed in position in the instrument. Light should be 
obtained by vaporization of a sodium salt in the flame. It is con- 
venient to employ a mixture made by fusing equal parts of sodium 
chloride and sodium phosphate, pulverizing the resulting mass. The 
handle of the instrument is rotated through 20 or 30 degrees to secure 
marked contrast between the two halves of the field and the eye-piece 
is focussed by drawing it out until a sharp line divides the two fields. 
The handle is then turned until the halves have the same intensity of 
brightness. Three or four readings should be made and the average 
taken, resting the eyes between readings. 

For computing the amount of glucose the following formula may be 
used, in which G represents the grams of glucose in 100 cc. of urine; A 
the angle of rotation and L the length of the tube employed: The 

A X 100 
52.5 X L 

tubes used for clinical purposes (polarizing saccharimeters) are made 
so that the readings may be made directly on the scale. With the 
short tube, 94.7 mm. long, each degree of rotation represents 2 per 
cent, of glucose, while with the long tube (189.4 mm. long) each degree 
represents 1 per cent, of glucose. 

If a solution of glucose be prepared for testing the instrument it 
must be allowed to stand overnight, since it is birotary when first 
dissolved. 

The specific rotation of the different sugars may be taken as dextrose, 
+52.5°; lactose, +53°; d-galactose, +81; levulose, -92°. 

Chlorides. — Simple Qualitative Test.— If albumin be present, it should 
be removed by boiling a portion of the urine after acidifying it with 
acetic acid, and by filtering through paper. About 10 cc. of the 
filtrate should be employed. This should be strongly acidulated with 
nitric acid to prevent precipitation of the phosphates by the silver. 
About 5 drops of a 10 per cent, solution of silver nitrate are added. A 
similar quantity of normal urine should be treated in the same way as 
a control. When chlorides are present in the usual quantity, a heavy 
curdy precipitate appears and settles to the bottom of the tube quite 
promptly. When the chlorides are diminished in quantity, the 
precipitate will be extremely light. This qualitative test is sufficient 
for the mere determination of an increase or diminution of the quantity 
of chlorides. 

Principle.— The chlorides are precipitated by silver nitrate, potas- 
sium chromate serving as an indicator of complete precipitation. 
When the chlorides have been precipitated completely, the silver 
nitrate is free to combine with the chromate radical, forming silver 
chromate, which is orange in color. 



250 EXAMINATION OF URINE 

Reagents Required.— (1) Standard Silver Nitrate Solution. — This 
should contain 29.059 gm. of pure silver nitrate dissolved in a liter of 
water. It should be prepared and standardized according to the 
method described in the following section. (2) Five per cent, potassium 
chromate. 

Procedure.— The urine is freed from albumin as described in the fore- 
going section (simple qualitative test). Ten cc. of the filtrate are 
placed in a porcelain evaporating dish with about 100 cc. of water. 
Five drops of 5 per cent, potassium chromate solution are added and 
the standard silver nitrate solution is run in from a burette until the 
color of the mixture in the evaporating dish becomes distinctly orange 
in color. The end-point is the first appearance of a definite and 
permanent orange-red. The mixture in the evaporating dish should 
be stirred constantly while adding the silver nitrate. A second 
determination should be made with a second 10 cc. quantity and the 
average of the two readings should be taken. 

Calculation.— One cc. of standard silver nitrate solution is precipi- 
tated by 0.01 gm. of sodium chloride. The number of cc. of silver 
solution required for neutralization of 10 cc. multiplied by 0.01 gives 
the chlorides in 10 cc. of urine. This result divided by 10 gives the 
amount per cc. 

Harvey's Modification of Volhard's Method.— Principle.— The chlo- 
rides are precipitated by a stipulated quantity of a standard solution 
of silver nitrate in the presence of nitric acid, and the amount of 
unprecipitated silver nitrate determined by titration with a standard 
solution of ammonium sulphocyanate, using a solution of iron alum 
as an indicator. When all of the silver has combined with the sulpho- 
cyanide to form silver sulphocyanate, additional ammonium sulpho- 
cyanate forms ferric sulphocyanate, which gives a yellow color and 
indicates the end-point. 

Reagents Required. — 1. Silver nitrate solution, of which 1 cc. 
precipitates 0.01 gm. of NaCl. For this 29.059 gm. of silver nitrate 
should be dissolved in 1 liter of water. This solution may be checked 
as follows: Chemically pure NaCl is dried at 120° C. for two hours. 
Then 0.150 gm. is weighed out and dissolved in 100 cc. of distilled 
water. Five drops of 5 per cent, potassium chromate are added and 
the silver solution is added from a burette. It should require 15 cc. 
of the latter to produce an orange tint. 

2. Ammonium sulphocyanate solution, of which 1 cc. precipitates 
1 cc. of the silver nitrate solution as described under 1. Since the salt 
absorbs water, it cannot be weighed with exactness. Thirteen grams 
are dissolved in 800 cc. of water. This solution is then titrated against 
the silver nitrate solution. Ten cc. of the silver nitrate solution are 
placed in an evaporating dish with 100 cc. of water. The mixture is 
acidified with HN0 3 and 2 cc. of the indicator solution are added. 
The ammonium sulphocyanate solution is then run in from a burette 
until a permanent red color is secured. If the solution be correctly 



QUANTITATIVE DETERMINATIONS 251 

prepared, exactly 10 cc. would be required. If less than this amount 
were needed, the solution was too strong and will require dilution with 
water; if more than 10 cc. were used, the solution was too weak and will 
require additional ammonium sulphocyanate. The exact amount 
may be computed mathematically, but after readjustment the solution 
must be titrated until its strength is such that exactly 10 cc. precipitate 
10 cc. of the silver solution. 

3. Indicator solution, made by dissolving 100 gm. of ferric ammonium 
sulphate in 100 cc. of 25 per cent, nitric acid. 

Procedure.— Measure 5 cc. of urine, freed from albumin, into a white 
porcelain evaporating dish. Add 20 cc. distilled water, 10 cc. standard 
silver solution, and 2 cc. of indicator solution. Mix thoroughly and 
run in standard ammonium sulphate solution from a burette until the 
first trace of yellow shows through the mixture. 

Calculation.— Subtract the number of cc. of sulphocyanate solution 
used in titration from 10 and multiply by 0.01 to determine the number 
of gm. of sodium chloride in 5 cc. urine, or by 0.20 to determine the gm. 
per 100 cc. 

Normal Quantity.— Under normal conditions, the amount of chlo- 
rides voided in twenty-four hours is between 10 and 15 gm. 

Significance.— The regulation of normal metabolism requires a 
chloride equilibrium, a definite amount being retained by the tissues 
of the body for the maintenance of osmotic equilibrium. Ingestion 
of greater amounts of chloride than usual, may be followed by tempor- 
ary retention but the excess is eliminated rather rapidly. Similarly 
a diminution in the intake of chlorides (as in starvation) results in a 
diminished chloride excretion, since the chlorides are held by the body 
to maintain a suitable chloride balance. The amount of chlorides 
excreted is diminished during the course of febrile diseases, where 
there are large exudates, in severe diarrhea, in pneumonia, and after 
inhalation of chloroform. The chloride excretion increases in amount 
during and after the crisis in pneumonia or when an exudate is absorbed. 
The chloride equilibrium is disturbed in advanced chronic nephritis 
with edema, the chlorides being retained. Attempts have been made 
to use chloride excretion as a prognostic guide in pneumonia, but 
without success. 

Determination of Phosphates and Sulphates.— The determination of 
these substances has value only in metabolic work, and methods for 
making these determinations may be obtained from standard texts 
on physiological chemistry. Determinations made on a single speci- 
men, without regard to the intake and to the analysis of feces, have no 
value whatever. The clinical value of the results of phosphate and 
sulphate determination, does not justify the description of the technic 
in this book. 

Total Nitrogen.— Principle.— By boiling urine with concentrated 
sulphuric acid in the presence of an oxidizing agent, the nitrogenous 
bodies are converted to ammonium sulphate. This is broken down 



252 EXAMINATION OF URINE 

by the action of an alkali with the formation of ammonia, which is 
liberated by distillation and is collected in acid of known strength. 
The acid may then be titrated with a standard alkali solution, and the 
amount of liberated and neutralized ammonia determined. 

Reagents Required. — (1) Crystals of copper sulphate. (2) Con- 
centrated sulphuric acid. (3) Potassium sulphate. (4) Powdered 
pumice. (5) Paraffin. (6) Decinormal sulphuric acid. (7) Congo- 
red solution. (8) Decinormal sodium hydrate. 

Procedure. — In a long-necked Kjeldahl flask should be placed 5 cc. 
of urine, measuring the quantity with volumetric pipette. A crystal 
of copper sulphate and 20 cc. of concentrated sulphuric acid should be 
added. The flask is heated over wire-gauze in a fume-closet until 




Fig. 71. — Kjeldahl distilling apparatus. A, flask containing digested urine; B, safety 
bulb; C, Liebig condenser; D, safety bulb; E, flask for receiving distillate. 



white fumes are driven off, when 2 gm. of potassium sulphate are added. 
This aids the process of decomposition by raising the boiling-point of 
the sulphuric acid. The mixture should be boiled for at least thirty 
minutes after it has become colorless. The flask is cooled and 300 cc. 
of ammonia-free distilled water are added, with a teaspoonful of 
powdered pumice to prevent bumping, a small piece of paraffin to pre- 
vent frothing, and 60 cc. of 30 per cent, sodium hydrate to alkalinize 
the mixture. More than this quantity of NaOH should be used if 
needed to accomplish the result. The alkali should not be allowed to 
touch the neck of the flask, since its presence here would interfere with 
the tight-fitting of the rubber stopper. The mixture is shaken and 
is ready for distillation (Fig. 71). The flask is connected with the 
safety-bulb of the apparatus, and the tip of the glass safety-tube as it 



QUANTITATIVE DETERMINATIONS 253 

emerges from the condenser is placed under the level of the liquid in 
an Erlenmeyer flask of 500 cc. capacity, in which should have been 
placed 50 cc. of decinormal sulphuric acid with ten drops of Congo-red 
solution and about 100 cc. of distilled water. Distillation should be 
conducted for at least an hour, starting with a small flame. At the 
end of this time, a drop of the distillate should be tested with litmus 
paper as it drops from the tip of the safety-tube. If still alkaline, 
distillation should be continued until the distillate no longer turns red 
litmus-paper blue. When the fluid from the condenser is no longer 
alkaline, boiling is discontinued and the fluid in the Erlenmeyer flask is 
titrated by adding decinormal sodium hydrate from a burette until the 
fluid shows a brilliant red color. 

When reagents of unknown quality are being employed, a blank 
determination should be run through, omitting only the urine, and 
the result obtained should be subtracted as a correction from that 
secured with regular determinations. 

Resume of Procedure, 
digestion. 

1. Place 5 cc. of urine in long-necked Kjeldahl flask. 

2. Add (a) 20 cc. of concentrated sulphuric acid. 

(b) A crystal of copper sulphate. . 

3. Heat until white fumes appear. 

4. Add 2 gm. of potassium sulphate. 

5. Boil for thirty minutes after mixture becomes colorless. 

6. Cool flask. 

DISTILLATION. 

7. Add (a) 300 cc. of distilled water. 

(b) Teaspoonful of powdered pumice. 

(c) Small piece of paraffin. 

(d) 60 cc. of 30 per cent, sodium hydrate, or more if needed 

to alkalinize. 

8. In Erlenmeyer flask place: 

(a) 50 cc. of T N o H 2 S0 4 . 

(b) 100 cc. of distilled water. 

(c) 10 drops of Congo-red solution. 

9. Start distillation with low flame, continuing for at least an hour, 

until the distillate fails to give alkaline reaction with litmus. 

TITRATION. 

10. Titrate contents of Erlenmeyer flask with .-jo NaOH until 

brilliant red color is obtained. 

11. Make calculation as shown below. 

Calculation.— Subtract the number of cubic centimeters of -^o 
NaOH used in titration from the number of cubic centimeters of j-q 



254 EXAMINATION OF URINE 

H2SO4 in the Erlenmeyer flask. The difference equals the number of 
cc. of Y^ H2SO4 neutralized by the liberated ammonia. Since 1 cc. 
of yyi H2SO4 is equivalent to 0.0014 gm. of nitrogen, the total amount 
of nitrogen is equal to the number of cc. of y^ H 2 S0 4 neutralized 
multiplied by 0.0014. The result represents the amount in 5 cc. of 
urine. The percentage can be ascertained by multiplying the number 
of cc. of ^ H2SO4 neutralized by the factor 0.028. 

Colorimetric Method (Myer's Modification of Folin's Method).— 
This method is to be given preference for clinical work. 

Principle.— A small quantity of urine is treated with sulphuric acid 
in the presence of an oxidizing agent and the nitrogenous bodies are 
converted to ammonium sulphate, as in the preceding method. After 
dilution the residue is Nesslerized and compared colorimetrically with 
a standard solution, similarly Nesslerized, containing a known quantity 
of nitrogen in the form of ammonium sulphate. 

Reagents Required.— {I) Crystals of copper sulphate. (2) Crystals 
of potassium sulphate. (3) Sulphuric acid (C. P.). (4) Hydrogen 
peroxide. (5) Standard solution of ammonium sulphate, 5 cc. con- 
taining 1 mg. of nitrogen (6) Nessler's solution. For the preparation 
of the two latter reagents, see pages 177 and 178. 

Procedure.— To 5 cc. of urine are added 45 cc. of water, making a 
1 : 10 dilution. One cc. of the diluted urine is placed in a thick-walled 
test-tube and to it are added one drop of 10 per cent, copper sulphate 
solution, a crystal of potassium sulphate, and 0.1 cc. of sulphuric acid, 
C. P. The tube is held in a clamp attached to a tripod and heat is 
applied with a micro-burner, first to remove the water and then to 
digest the nitrogenous compounds with the sulphuric acid. Provision 
must be made for carrying off the fumes (Fig. 56). Heating is con- 
tinued for two minutes after the mixture becomes colorless. If a clear 
blue color does not develop readily, a drop or two of hydrogen peroxide 
is added while the mixture is still warm. It is then allowed to cool 
somewhat though not to the point of solidification. 

Two 100 cc. cylindrical graduates should be in readiness. A 20 per 
cent, dilution of Nessler's solution is made up with distilled water. 
The contents of the tube are dissolved quickly in about 10 cc. of water 
and the solution is poured into the graduate, rinsing the tube into the 
graduate with a little distilled water to bring the total volume to 15 cc. 
In the second 100 cc. graduate is placed 5 cc. of the standard solution 
of ammonium sulphate (5 cc. of which contains 1 mg. of nitrogen) and to 
it is added 20 cc. of 20 per cent. Nessler's solution and distilled water to 
bring the volume up to 100 cc. To the unknown, a sufficient quantity 
of the 20 per cent. Nessler's solution is added quickly to secure the 
maximum amount of red coloration, when the mixture is diluted with 
water to make the total volume either 50 or 100 cc, whichever amount 
is required to make the color of the diluted unknown most closely 
approximate the color of the standard. A portion of the standard is 
then placed in the left-hand cup of the colorimeter and the cup is set 



QUANTITATIVE DETERMINATIONS 255 

at a depth of 10 mm.; this is moved up and down until the color in the 
two halves of the visual field match. 



Resume of Procedure. 

1. Make a 1 : 10 dilution of urine. 

2. Place 1 cc. of dilution in a thick- walled test-tube. 

3. Add the following: 

(a) 1 drop of 10 per cent, copper sulphate. 

(b) Crystal of potassium sulphate. 

(c) 6 drops of sulphuric acid, C. P. 

4. Place tube in clamp and heat with micro-burner until mixture 

becomes colorless. 

5. If blue color does not appear, add a drop or two of hydrogen 

peroxide. 

6. Cool, but not to the point of solidification. 

7. Take two graduated cylinders of 100 cc. capacity and handle as 

follows, placing digested urine mixture in cylinder B. 

CYLINDER A. CYLINDER B. 

8. Place in cylinder 5 cc. of Digest mixture with 10 cc. of 

standard ammonium sul- water, 

phate solution, containing 
1 mg. of nitrogen. 

9. Add 20 cc. of 20 per cent. Add 20 per cent. Nessler's solu- 

Nessler's solution. tion to secure maximum color. 

10. Add water to make total vol- Add water to make color approxi- 

ume 100 cc. mately same as solution in A. 
11. Compare in colorimeter. 

Calculation. — Let R represent the reading of the unknown when the 
standard is set at ten. When the unknown is diluted to 100 the number 
of milligrams of nitrogen per cc. of undiluted urine can be ascertained 

by the following equation : Mg. nitrogen per cc. = -p- " If the unknown 

were diluted to only 50 cc. the result should be divided by 2. The 
same figure also represents the gms. per liter. The percentage is 
represented by placing a decimal point in front of the figure ascer- 
tained. 

Normal Quantity.— In health on an average diet the total amount of 
nitrogen excreted in the urine in twenty-four hours is about 16 to 18 
gm. 

Urea.— Principle.— Urea is converted to ammonium carbonate by 
urease, an enzyme found in certain fungi, bacteria, and higher forms of 
plant life. By comparing the alkalinity of urine treated with urease 
with that of an untreated specimen of equal volume, the amount of 



256 EXAMINATION OF URINE 

urea may be computed. Urease for biochemical purposes is ordinarily 
obtained from the soy bean (glycerine hispida) or from the jack bean. 

Urease.— It is desirable to obtain urease in the form of a dry and 
readily soluble powder, prepared with potassium dihydrogen phosphate 
and dipotassium hydrogen phosphate so combined that the mixture 
will be exactly neutral on solution. The neutral phosphate mixture 
serves as a stabilizer for the enzymes. Urease in this form is prepared 
by the Arlington Chemical Company, Yonkers, N. Y., and by E. R. 
Squibb and Sons. Compressed tablets of urease are put on the market 
by Hynson, Westcott and Dunning, Baltimore, Md. The activity of a 
particular lot of urease should be determined. Usually a two and one- 
half to five per cent., of the Squibb preparation and a ten per cent, 
solution of the Arlington preparation may be made. 

Three methods employing this principle are available. The extreme 
simplicity of Marshall's' method commends it to the practitioner. 

Determination by Direct Titration of Urine Treated with Urease 
(Marshall's Method).— Procedure— This is the simplest method. 
Two Erlenmeyer flasks of about 200 cc. capacity are taken. Into each 
is placed 1 cc. of the urine to be examined. In' one flask is placed 
urease, either 2 cc. of the freshly prepared solution or two tablets 
(as prepared by Hynson, Westcott and Dunning). The latter should 
be thoroughly crushed with a little water before being added. Enough 
distilled water should be added to each flask to bring the total volume 
up to about 100 cc. The two flasks should then be subjected to a 
temperature of 40° C. for fifteen minutes, when into each flask should 
be placed 5 drops of 1 per cent, alcoholic solution of methyl orange. 
^ HC1 is added from a burette until the color becomes distinctly pink. 

Calculation.— The amount of ammonium carbonate formed is indi- 
cated by the increased alkalinity of the specimen treated with urease 
compared to the control specimen. From the number of cc. of ^ HC1 
required to neutralize the specimen treated with urease, subtract the 
number of cc. required to neutralize the control, and multiply the 
remainder by the factor 0.6. The result will give the number of gm. of 
urea per 1000 cc. of urine. The amount of urea nitrogen may be 
computed by dividing this figure by the factor 2.14. 

Determination by Aspiration and Titration of the Liberated Ammonia 
after Treatment of the Urine with Urease (Van Slyke and Cullen).— 
Urine is treated with urease as in the Marshall method and the ammon- 
ium carbonate is broken down to ammonia by the addition of a strong 
alkali. The liberated ammonia is then aspirated by an air current 
into a measured quantity of a standard acid solution, by which it is 
neutralized. The amount so liberated and neutralized may be deter- 
mined by titrating the acid with a standard alkaline solution. 

In this method, the urinary ammonia is determined as well as the 
amount of ammonia formed from urea. It is necessary, therefore, to 
ascertain the amount of urinary ammonia by the same method, treating 
a similar quantity of urine with alkali and aerating but omitting the 



QUANTITATIVE DETERMINATIONS 257 

treatment with urease. The amount of urinary ammonia is sub- 
tracted from the amount of urinary ammonia plus urea to give the 
amount of urea. 

Reagents Required.— Solution of urease: Amyl or caprylic alcohol: 
t^ HC1; 1 per cent, solution of sodium alizarinate; -^ sodium hydrate; 
saturated solution of sodium carbonate. 

Procedure.— The urine is diluted by adding 45 cc. of water to 5 cc. 
of urine. Into cylinder A of the aerating device (Fig. 55) there should 
be measured 5 cc. of the diluted urine, 2 cc. of urease solution, and a 
drop of caprylic or amyl alcohol to prevent bumping. The stopper 
is closed and the cylinder is placed in a water-bath at a temperature of 
40° C. for fifteen minutes to allow the enzyme to act. Meanwhile 
25 cc. of ^ HO (or H2SO4) are placed in cylinder B with one drop of 
1 per cent, sodium alizarinate indicator and one drop of caprylic or 
amyl alcohol. When fifteen minutes have expired, A and B in series 
are connected and the air current B allowed to pass for a half minute, 
in order to drive over into the acid in B any ammonia which may have 
escaped into A. The air should be drawn through a wash-bottle 
containing dilute sulphuric acid to remove any contained ammonia. 
At the end of the preliminary aspiration, cylinder A is opened and 5 cc. 
of a saturated solution of sodium carbonate are added. A is again 
closed and the air current is allowed to pass until all the ammonia is 
exhausted from A. The time required will vary with the efficiency of 
the pump or pressure system which is used, ranging from five to thirty 
minutes. When aspiration is completed, the tube in B is washed into 
B with a small quantity of distilled water and -^ sodium hydrate is 
added to the contents of B until a purple color appears. 

Resume of Method. 

1. Make a 1 : 10 dilution of urine. 

2. Place 5 cc. of this dilution in cylinder A. 

3. Add 

(a) 2 cc. of 20 per cent, urease solution. 

(b) A drop or two of amyl or caprylic alcohol. 

4. Stopper A and place in water-bath at 40° C. for fifteen minutes. 

5. In B place 

(a) 25 cc. of /o HC1. 

(5) A drop of amyl or caprylic alcohol. 

(c) A drop of 1 per cent, sodium alizarinate. 

6. Connect A and B in series, turn on air current and allow it to 

pass about one-half minute. 

7. Disconnect, add 5 cc. of saturated solution sodium carbonate to A. 

8. Reconnect A and B and allow air current to pass about thirty 

minutes. 

9. Wash absorption tube in B into cylinder B with water. 

10. Titrate contents of B using -^ NaOH until purple color appears. 

11. Make calculation as shown below. 
17 



258 EXAMINATION OF URINE 

Calculation.— The number of cc. of fiftieth-normal acid neutralized 
by the liberated ammonia is computed by subtracting from 25 the 
number of cc. ^ sodium hydrate used in the titration. This is multi- 
plied by the factor 0.56 to give the number of gm. of urea plus ammonia 
nitrogen in 1000 cc. of urine. To ascertain the number of gm. urea 
nitrogen, determine the amount of ammonia nitrogen and subtract. 
The amount of urea may be calculated from the urea nitrogen by 
multiplying by the factor 2.14. 

Myers' Colorimetric Method.— This is the preferred method. 

Reagents Required.— (1) Solution of urease. (2) Amyl or caprylic 
alcohol. (3) ^ HC1. (4) Saturated solution of sodium carbonate. 
(5) Nessler's solution. (6) Standard solution of ammonium sulphate, 
5 cc. containing 1 mg. of nitrogen. (For the preparation of the latter 
two reagents, pages 177 and 178.) 

Procedure.— Two cc. of a 1 : 10 dilution of urine are placed in a 
test-tube with 1 cc. of urease solution (10 per cent, in strength if the 
Arlington brand be employed) and a drop of two of amyl or caprylic 
alcohol. The tube is stoppered and placed in a water-bath at 40° C. 
for fifteen minutes as in the preceding method. Aeration is conducted 
also as was directed before, the test-tube being placed in cylinder A, 
cylinder B being charged with 25 cc. of ^r HC1 acid (Fig. 55) . Before 
the air current is started, 5 cc. of a saturated solution of sodium car- 
bonate should be added to the contents of the tube in cylinder A. 
Aeration is allowed to proceed for about thirty minutes. The contents 
of the test-tube are placed in a 100 cc. graduated cylinder. The tube 
is rinsed with a few cc. of distilled water which are poured into the 
graduate also, and freshly diluted 20 per cent. Nessler's solution is 
added until there is no further deepening of the color. Distilled water 
is added to make the total volume 50, 100, or 200 cc, whichever may 
be necessary to most nearly approximate the color of the standard. 

The standard is prepared simultaneously by putting in a similar 
cylindrical graduate 5 cc. of the ammonium sulphate standard (5 cc. 
containing 1 mg. of nitrogen), 10 cc. of water, 20 cc. of the freshly diluted 
20 per cent. Nessler's solution, and distilled water in sufficient quantity 
to make the total volume 100 cc. 



Resume of Method. 

1. Make a 1 : 10 dilution of urine. 

2. Place 2 cc. in a test-tube. 

3. Add 

(a) 1 cc. of urease solution. 

(b) A drop or two of amyl or caprylic alcohol. 

4. Stopper tube and place in water-bath at 40° C. for fifteen minutes. 

5. In cylinder B place: 

(a) 25 cc. of & HC1. 

(b) A drop or two of amyl or caprylic alcohol. 



QUANTITATIVE DETERMINATIONS 259 

6. Place test-tube in cylinder A and remove stopper. 

7. Add 5 cc. saturated solution of sodium carbonate. 

8. Connect cylinders A and B, turn on air-current. Allow air to 

pass for about thirty minutes. 

9. Wash out absorption tube in B into cylinder B with 2 or 3 cc. of 

water. 

10. Treat contents of cylinder B as shown below, making up a standard 

for comparison in cylinder C. 

CYLINDER B. CYLINDER C. 

11. Place in cylinder 5 cc. of ammo- 

nium sulphate standard (con- 
tains 1 mg. of nitrogen). 

12. Add 20 per cent, dilution of Add 20 cc. of 20 per cent, solution 

Nessler's solution until no of Nessler's solution, 

further deepening of color. 

13. Add distilled water to 50, Add distilled water to make 

100, etc., cc, to match volume 100 cc. 

color of C approximately. 

14. Compare in colorimeter. 

Calculation.— The results may be arrived at by dividing 50 by the 
reading when the standard is set at 10. If the dilution were made to 
100, the result is the number of gm. of urea and ammonia nitrogen per 
cc. of urine. If the dilution were to 50, the result should be divided by 
2, and if to 200, it should be multiplied by 2. 

Normal Quantity.— The amount of urea nitrogen excreted in twenty- 
four hours by a normal individual on an average diet is 13 to 16 gm. 
Of the total nitrogen excreted, from 86 to 90 per cent, is urea nitrogen. 

Ammonia Nitrogen. — The determination of ammonia may be per- 
formed at the same time that the urea determination is made, setting 
up the two cylinders necessary for aspiration in series with those used 
for the urea determination. When the amount of ammonia nitrogen 
has been ascertained, it may be subtracted from the amount of urea and 
ammonia nitrogen, and the remainder will represent the urea nitrogen. 
This may be translated to terms of urea by multiplying by the factor 
2.14. 

Ammonia.— Determination by Aspiration and Titration (Folin's 
Method.)— This is the simpler method. 

Reagents Required. — See determination of urea by aspiration and 
titration. Urease is not required. 

Principle.— The principle and procedure is the same as that for the 
determination of urea by aspiration and titration except that urease 
is not used. Five cc. of urine are placed in cylinder A (Fig. 55) of the 
aspiration cylinder, with a drop or two of amyl or caprylic alcohol. 
In cylinder B should be placed 25 cc. of ^ HC1 (or H 2 S0 4 ), with a drop 



260 EXAMINATION OF URINE 

of 1 per cent, sodium alizarinate and a drop or-two of amyl or caprylic 
alcohol. In cylinder A are put 5 cc. of a saturated solution of sodium 
carbonate. The cylinders are connected and the air current is 
allowed to pass through for the requisite length of time. For details 
regarding aeration, see the application of the same method to the 
determination of urea and ammonia nitrogen in the preceding para- 
graphs. When the aeration is complete, the absorption-tube in B is 
washed into B with a little distilled water and X^ XaOH is added 
from a burette to the contents of B till a purple color appears. 

Calculation.— From 25 is subtracted the number of cc. of ^~ XaOH 
required to neutralize the acid. The difference represents the number 
of cc. of acid neutralized by liberated ammonia. Multiplying this by 
the factor 0.056 will give the number of grams of ammonia nitrogen per 
1000 cc. of urine. The ammonia may be determined by multiplying 
the ammonia nitrogen by the factor 1.21. 

Colon 'metric Method. — Reagents Required.— As for the determination 
of urea by the colorimetric method, except that urease is not needed. 

Procedure.— Undiluted urine is used. The determination is per- 
formed in the same manner as described for the determination of urea 
and ammonia nitrogen in the preceding section (see page 258) except 
of course that urease is not added to break down urea into ammonium 
carbonate. Two cc. of undiluted urine are placed in cylinder A of 
the aeration outfit and cylinder B is charged with 25 cc. -^ HC1. 
Five cc. of a saturated solution of sodium carbonate are added to the 
urine in test-tube A, which is then placed in the cylinder, the cylinders 
are stoppered, and the air current allowed to run through until the 
ammonia has been completely removed. Xesslerization is conducted 
as described in the preceding section for the determination of urea 
and ammonia nitrogen, and the colorimetric reading and calculation 
is made in the same way. The result gives the number of grams of 
ammonia nitrogen per cc. of urine. Multiplying this by the factor 
1.21 will give the number of grams of ammonia per cc. of urine. 

Folin's Alternate Clinical Method. — Principle.— The acidity of a 
given quantity of urine is determined by titration with fV XaOH. To 
the urine is then added neutralized formalin, which combines with the 
ammonia to form neutral hexamethylene-tetramin and so sets free the 
acid equivalent of the amount of ammonia present. 

Reagents Required.— {I) j n XaOH solution. (2) phenolphthalein 
solution. (3 ) a saturated solution of potassium oxalate solution (neutral 
to phenolphthalein). (4) formalin, also neutral to phenolphthalein. 

The two latter reagents are neutralized by adding a few drops of the 
phenolphthalein solution and then enough j-g XaOH to produce a faint 
pink coloration. 

Procedure.— Place in a flask 5 cc. of the neutralized potassium oxalate 
solution, 3 drops of phenolphthalein solution, and 25 cc. of urine. 
Titrate with , N U XaOH to produce a faint but unmistakable pink color. 
Then add 5 cc. of neutralized formalin and again titrate to the same 



QUANTITATIVE DETERMINATIONS 261 

pink color. The difference between the total amount of alkali employed 
and that used for the first titration represents the alkali required to 
neutralize the acid set free by the combination of ammonia with 
formaldehyde. Each cc. of y^ alkali used corresponds to a cc. of" 
yq ammonia. 

Calculation. — This should be carried out as in the previous test. 

Normal Quantity.— In health with an average diet the amount of 
ammonia nitrogen excreted in twenty-four hours is about 0.7 gm. 
The ammonia nitrogen represents from 3.3 to 5 per cent, of the total 
nitrogen excreted. The amount is greatly increased in acetone acidosis. 

Determination of Uric Acid. — Colorimetric Method.— (Benedict and 
Hitchcock's modification of Folin and Denis' method.) 

Principle. — The purins are precipitated by an ammoniacal silver- 
magnesia mixture. The precipitate is separated by removing the 
ammonia and then is dissolved with hydrocyanic acid. In the presence 
of the phosphotungstic acid of the Folin-Denis reagent a color reaction 
is obtained, which may be used as a quantitative test by employing a 
comparison tube containing a known quantity of uric acid, similarly 
treated. 

Reagents Required.— (1) Saturated solution of sodium carbonate. 
(2) 5 per cent, solution of sodium or potassium cyanide. (3) Folin-Denis 
reagent (for preparation of this reagent, see page 191). (4) ammonia- 
cal silver-magnesium mixture. 

For this it is necessary to make a magnesia mixture first, which 
is prepared by dissolving 36 gm. of magnesium sulphate and 70 gm. 
of ammonium chloride in 280 cc. of distilled water and then adding 
140 cc. of concentrated ammonia. 

The ammoniacal silver-magnesia solution is prepared according to 
the following formula : 

3 per cent, silver nitrate solution 70 cc. 

Magnesia mixture 30 " 

Concentrated ammonia 100 " 

If turbidity develops, the mixture should be filtered. 

Procedure.— In a graduated 15 cc. centrifuge tube with conical tip 
(Fig. 54, g) are placed 2 cc. of urine and 3 cc. of water. To this are 
added 15 drops of silver magnesium mixture. To cause complete 
precipitation of the purins, the tube is placed in a beaker containing 
cracked ice and salt, where it is allowed to remain for fifteen minutes. 
It is then centrifugalized and the supernatant fluid is decanted off. 
The tube is stood upside down to drain off the residual fluid and the 
ammonia is gotten rid of by introducing well into the tube a piece of 
glass tubing attached to a suction pump. 

To the precipitate in the tube are added 2 or 3 cc. of water and 2 
drops of a 5 per cent, solution of potassium (or sodium) cyanide. A fine 
glass rod should be used to stir the sediment and to secure complete 
solution. Two cc. of Folin-Denis reagent are then introduced and the 



262 



EXAMINATION OF URINE 



contents of the tube are poured into a 100 cc. cylindrical graduate, 
rinsing the centrifuge tube into the graduate with a saturated solution 
of sodium carbonate, about 20 cc. carbonate solution are employed. 

The standard is prepared by placing in a similar cylindrical graduate 
of 100 cc. capacity, 5 cc. of the uric acid standard (prepared so that it 
will contain 1 mg. of uric acid), 2 drops of a 5 per cent, solution of 
potassium (or sodium) cyanide, 2 cc. of Folin-Denis reagent, 20 cc. 
of saturated solution sodium carbonate, and distilled water to make the 
total volume 100. Water should now be added to the graduate con- 
taining the unknown to 50, 100, or even 200 cc, as may be necessary 
to make the colors in the two graduates of approximately the same 
depth. In making up the standard and the unknown it is well to carry 
out the various steps as nearly simultaneously as possible. 

Resume of Method. 

1 . Place 2 cc. of urine in graduated centrifuge tube. 

2. Add (a) 3 cc. of distilled water. 

(6) 15 drops of ammoniacal silver magnesia mixtures. 

3. Stand tube in mixture of cracked ice and salt for fifteen minutes. 

4. Centrifugalize. 

5. Decant off supernatant fluid and discard it. 

6. Drain fluid from tube and aspirate the ammonia. 

7. Treat precipitate of uric acid in tube as shown below, preparing 

standard at the same time. 



UNKNOWN. 

8. Add 3 cc. water to sediment in 
the tube. 



9. Add 2 drops of 5 per cent, 
sodium cyanide solution. 

10. Add 2 cc. of Folin-Denis re- 

agent. 

11. Pour into 100 cc. cylinder and 

rinse tube into cylinder with 
saturated solution of 20 cc. 
of sodium carbonate. 

12. Add water to 50, 100, or 200 cc. 

mark to approximately 
match color of standard. 



STANDARD. 

Place in 100 cc. cylinder 5 cc. 
of uric acid standard solution 
containing 1 mg. of uric acid 
nitrogen. 

Add 2 drops of 5 per cent, 
sodium cyanide solution. 

Add 2 cc. of Folin-Denis re- 
agent. 

Add 20 cc. saturated solution 
of sodium carbonate. 



Add water to make volume 
100 cc. 



13. Compare contents of two cylinders in colorimeter. 



Calculation.— When the unknown is diluted to 100 cc, and the cup 
of the colorimeter containing the standard is set at ten, the figure 5 
may be divided by the reading of the unknown. This will give the 
number of gm. of uric acid nitrogen in 1000 cc. of urine. 



QUANTITATIVE DETERMINATIONS 263 

When the unknown has been diluted to 50 cc, the same formula may 
be used, but the final result should be divided by 2, while if it were 
diluted to 200, the result should be multiplied by 2. 

Normal Findings.— Under conditions of health with an average diet, 
the amount of uric acid nitrogen ranges from 0.08 to 0.15 gm. per diem. 
The average output makes up from 0.6 to 1.0 per cent, of the total 
nitrogen. 

Determination of Creatine and Creatinine (Folin's later method, 
Folin and Morris) . 

Principle.— Folin has raised the objection that his older colori- 
metric method, which employed a bichromate standard, imposed an 
unnecessary limitation upon a flexible method since the results were 
unsatisfactory with small amounts of creatinine. He therefore, 
modified this method so as to employ a creatinine standard, the urine 
and the standard being subjected to the same treatment with alkali 
in the presence of picric acid. The principle is the same as that under- 
lying the determination of creatinine in the blood, relying upon the 
power of creatinine to reduce sodium picrate. 

Reagents Required.— (1) Standard creatinine solution, containing 
1 mg. of creatinine in 1 cc. of a saturated solution of picric acid (see 
page 444). (2) 10 per cent, solution of sodium hydrate. 

Procedure for Creatinine. — One cc. of the standard creatinine solu- 
tion (containing 1 mg. of creatinine) is placed in a 100 cc. volumetric 
flask, and 1 cc. of urine is placed in another 100 cc. volumetric flask. 
To each flask are then added 20 cc. of saturated picric acid solution 
measured with a volumetric pipette, and 1.5 cc. of 10 per cent, sodium 
hydrate solution, measured with a burette or with a pipette graduated 
in 0.1 cc. At the end of ten minutes the flasks are filled to the 100 cc. 
mark with distilled water. 

Resume of Method, 
flask a. flask b. 

1. Place in flask 1 cc. of urine. Place in flask 1 cc. of standard 

creatinine solution (1 mg. 
of creatinine) . 

2. Add 20 cc. of saturated solution Add 20 cc. of saturated solution 

of picric acid. of picric acid. 

3. Add 1.5 cc. of 10 per cent solu- Add 1.5 cc. of 10 per cent solu- 

tion of sodium hydrate. tion of sodium hydrate. 

4. Allow to stand for ten minutes. Allow to stand for ten minutes. 

5. Add distilled water to 100 cc. Add distilled water to 100 cc. 

mark. mark. 

6. Compare a portion of contents of two flasks in colorimeter. 

Calculation.— When the standard has been set at 10 mm., ten is 
divided by the reading of the unknown. The results give the mg. of 
creatinine in the volume of urine employed in making the determina- 



264 EXAMINATION OF URINE 

tion. If the urine reads less than two-thirds or more than one and a 
half that of the standard, the determination should be repeated with 
more or with less urine, as the case may be, so that the amount of 
creatinine in the unknown will be approximately that in the standard. 

Procedure for Creatine.— Creatine is broken down to creatinine by 
prolonged boiling with picric acid and the total creatinine is then 
determined. From this the amount of preformed creatinine, which 
has been determined previously, may be subtracted. 

Into a weighed FMenmeyer Pyrex flask of 200 cc. capacity is placed 
enough urine to yield about 0.7 to 1.5 mgm. of creatinine when the 
creatine is broken down. With this is placed 20 cc. of a saturated solu- 
tion of picric acid and 130 cc. of distilled water. A few small pebbles 
are used to prevent bumping and the mixture is boiled gently over a 
microburner for about an hour. Toward the end of the time, the heat 
is increased so that the solution may be boiled down to somewhat less 
than 20 cc. The flask is placed upon the scales and enough water 
added to bring the w r eight of the contained fluid to 20 to 25 gm. The 
flask and its contents are then cooled in running water and 1.5 cc. of 
10 per cent. NaOH are added, the total volume is brought up to 100 cc. 
with distilled water, and the amount of creatinine is determined by using 
a standard of known strength, as described in the previous section. 

Having determined the amount of preformed creatinine, this may be 
subtracted from the result just obtained to give the amount of creatine. 

Folin's Original Method. — This method is given because it is still 
preferred by many workers. A solution of potassium bichromate 
solution serves as a standard. 

Creatinine. — Place 10 cc. of urine in a 500 cc. volumetric flask and 
add 15 cc. of a saturated solution of picric acid and 5 cc. of 10 per cent, 
solution of sodium hydrate. Shake the mixture thoroughly and allow 
it to stand for five minutes. Then fill the flask to the 500 cc. mark and 
after mixing, pour a portion into one of the cups of the colorimeter. 
Set the other cup, filled with § potassium bichromate solution (24.55 
gm. to the liter) at the 8 mm. mark. Take the average of several read- 
ings. 8.1 divided by the figure obtained multiplied by the total volume 
of urine gives the daily output of creatinine. 

Creatine (Folin-Benedict and Myers' method). — Pipette two 10 cc. 
portions of urine into two 100 cc. Erlenmeyer flasks, and add to each 
flask 10 cc. of normal hydrochloric acid. Heat the flasks in the auto- 
clave at 20 pounds pressure for thirty minutes to convert creatine to 
creatinine. Then use the contents for creatinine determinations as 
given above, adding to each flask 10 cc. of 10 per cent, sodium hydrate 
using 10 cc. instead of 5 cc. to overcome the increased acidity. The 
result represents creatine and creatinine. Creatine may be obtained 
by subtracting the amount of preformed creatinine. 

Normal Quantity.— The normal output of creatinine nitrogen on a 
standard diet is from 0.50 to 0.(50 gm. per day. The creatinine nitrogen 
makes up from 3.2 to 4.5 per cent, of the total nitrogen. 



CHEMICAL COMPOSITION OF NORMAL URINE 265 

NITROGEN PARTITION. 

The principal non-protein nitrogenous constituents of the urine are 
urea, ammonia, creatinine, and uric acid, which ordinarily compose 
about 95 per cent, of the urinary nitrogen. The remainder is composed 
of small amounts of creatine, purine bases, pigments, amino-acids, 
hippuric acid, etc. Ordinarily the factors considered as of clinical 
importance are total nitrogen, urea nitrogen, and ammonia nitrogen. 
The amount of nitrogen excreted in various forms is determined, and 
the total nitrogen is ascertained. With these figures, percentages 
may be computed. Suppose, for example, the total nitrogen be 9.7 gm., 
the urea nitrogen 8.4 gm., the ammonia nitrogen 0.26 gm., and the 
creatinine nitrogen 0.64 gm. Then the distribution would be as 
follows : 

9.7 = 86.6 per cent. 



Urea nitrogen = 8.40 

Ammonia nitrogen = 0.26 



Creatinine nitrogen = 0.64 -¥■ 9.7 = 6.6 " 

The amounts of all these substances should be reduced to terms of 
nitrogen before the computations are made. 

CHEMICAL COMPOSITION OF NORMAL URINE. 

Folin has made practically complete examinations of thirty urines 
obtained from six individuals who were fed upon a so-called standard 
diet which contained the amount of protein demanded by Voit for 
individuals of their body weight. The diet contained about 119 gm. 
of protein and approximately 148 gm. of fat and 225 gm. of carbo- 
hydrates. The average weight of the individuals was 63.4 kilos., rang- 
ing from 56.6 to 70.9 kilos. The results were as follows: 

Final average, Minimum, Maximum, 

gm. gm. gm. 

Ammonia nitrogen 0.70 0.55 0.85 

Total nitrogen 16.00 14.80 18.20 

Urea nitrogen 13.90 12.80 16.20 

Creatinine nitrogen 0.58 0.50 0.66 

Uric acid nitrogen 0.12 0.08 0.15 

Undetermined nitrogen . . . 0.60 0.41 0.85 

Total sulphur as S0 3 .... 3.31 3.11 3.73 

Inorganic SOs 2.92 2.67 3.25 

Ethereal S0 3 0.22 0.19 0.25 

Neutral SO3 . 0.17 0.13 0.19 

Total phosphates as P2O5 ...3.87 3.44 4.50 

Chlorine 6.10 5.60 6.90 

Volume of urine, twenty-four-hour 

amount 1430 cc. 1196 cc. 1812 cc. 

The nitrogenous constituents may be expressed in terms of per- 
centage with regard to the total nitrogen content. 





Final average 


Minimum 


Maximum 




per cent. 


per cent. 


per cent. 


Urea 


. . . . 87.50 


86.20 


89.40 


Ammonia 


. . . . 4.30 


3.30 


5.00 


Creatinine 


. . . . 3.60 


3.20 


4.50 


Uric acid .... 


. . . . 0.80 


0.60 


1.00 


Undetermined 


. . . . 3.75 


2.70 


5.30 



266 



EXAMINATION OF URINE 



DETECTION OF MERCURY IN THE URINE. 

The exact diagnosis of mercury poisoning is of importance on account 
of the slow development of symptoms and the efficiency of modern 
treatment when instituted early, as has been shown by the work of 
Lambert and Patterson. 

Reagents Required.— (1) Hydrochloric acid, C. P. (2) potassium 
chlorate dry. (3) copper wire, 18 gauge (Brown and Sharpe wire 
gauge). (4) dentist'sgold foil; J. M. Ney'sgold velvet cylinder, cohesive, 
No. | is suitable for this purpose. 

Procedure. — (Method of Yogel and Lee.) About 150 cc. of the urine 
are placed in a beaker, acidulated with about 5 cc. of HC1 (C. P.), and 
boiled over a free flame until the bulk has been reduced to about 25 



\ \ 




Fig. 72. — Kjeldahl's distilling apparatus for a number of specimens. 



to 30 cc, when 2 to 3 cc. HC1 are added to restore the loss by evapora- 
tion. Then sufficient dry potassium chlorate is stirred in to render the 
mixture practically colorless, and about 30 cc. of distilled water are 
added. The mixture is boiled again over the free flame to drive off 
the chlorine until its odor cannot be detected. A piece of copper wire, 
about 4 cm. long, is bent twice upon itself and is cleaned by boiling for 
a few moments in a test-tube with a little dilute HO which may be 
poured off and replaced with two or three changes of distilled water. 
The wire is then dropped from the test-tube directly into the beaker of 
evaporated urine. It is allowed to remain here for two or three hours, 
or if no deposit be apparent on the wire, for a longer period of time, 
when it is transferred with a glass rod and carefully washed with 
a little distilled water. After gentle drying with filter paper, it is 
slipped into a piece of glass tubing (about 3 or 4 mm. diameter and 



TESTS FOR DETERMINATION OF RENAL FUNCTION 267 

15 cm. long) with one end sealed. The wire should not be touched with 
the fingers. It is then pushed down to the closed end of the tube. 
After it is inserted a small piece of dentist's gold foil, to occupy a position 
about 2 cm. above the upper end of the bent copper wire. The tube is 
held in a horizontal position and the end holding the wire is heated 
intermittently and gently over a low flame, preferably that of a spirit 
lamp (Fig. 73). Only the wire and not the foil should be heated. 
The gold foil should be watched carefully during this process for any 
appearance of silvering. When only a small quantity of mercury is 
present, there may be merely tiny silvery spots visible only with a 



Fig. 73. — Vogel and Lee's test for mercury, a, copper wire; b, flame of alcohol 
lamp; c, gold foil in capillary glass tube. 

magnifying glass, while with larger quantities there will be a readily 
perceptible amalgam. After the test has been completed, the open 
end of the tube may be sealed and the tube kept as a permanent record. 
Vogel and Lee state that the test is given with a dilution of mercuric 
chloride as low as 1 to 1,000,000, containing 0.00002 gm. of mercury. 
In stomach contents and urine, it was found that 1 mg. in 100 cc, 
equaling a dilution of 1 : 100,000, could be detected by allowing the 
copper wire to remain in the fluid two hours. Before attaching diag- 
nostic significance to the results, inquiry should be made as to taking of 
calomel, since it has been found that a positive test will be secured 
after ingestion of only 2 grains. 

TESTS FOR THE DETERMINATION OF RENAL FUNCTION. 

It is well recognized that there is by no means an invariable rela- 
tionship between the evidences of renal tissue damage such as the 
quantity of albumin and the number and type of tube casts on the one 
hand and the excretory capacity of the kidneys on the other. Further- 
more, it is evident that a determination of the constituents of the urine 
with the object of detecting an accumulation of waste products by the 
body can have a narrow range of usefulness unless there is a definite 
knowledge both of the intake of food materials and the determination 
of excretion by other channels such as the feces, perspiration, and 
respiration. Complete metabolic studies of this type are manifestly 
out of the question in ordinary clinical work, and are not always 



268 EXAMINATION OF URINE 

readily accomplished for clinical purposes even in well equipped 
hospitals. 

The introduction by Folin of colorimetric methods for blood analysis 
has placed at the disposal of the clinician a means of studying the 
chemical composition of the blood and of ascertaining whether there 
be an accumulation of waste products. The information so derived, 
correlated with the bedside findings and properly interpreted, is of 
great assistance in evaluating the functional power of the kidneys. 

Of methods which have been offered as tests for the functional power 
of the kidneys, two stand out as of the greatest clinical value. The 
first is the determination of the ability of the kidney to excrete phenol- 
sulphonephthalein. Numerous tests employing as their basic principle 
the excretion of dye substances have been proposed, but phenol- 
sulphonephthalein seems best adapted to practical usage. The second 
is the so-called " test-meal for renal function" proposed by Mosenthal, 
who adopted the suggestion of earlier workers that the response of the 
kidney to a full meal containing a reasonable amount of fluids, purins, 
and salts, varied in health and disease, and formulated a method 
whereby this might be studied by having the urine voided at fixed 
intervals after such a meal. 

As Macleod remarks, while the so-called renal tests are not free from 
criticism, they have contributed very useful information ; the excretion 
by the kidney of a chronic nephritic of a urine of more or less fixed 
specific gravity would suggest an impairment of the resorbing mechan- 
ism, and the failure of a kidney to excrete the proper amount of dye 
suggests an impairment in the filtering apparatus, but hard and fast 
rules cannot be applied and probably the tests must be interpreted at 
present for the kidney as a whole. 

Excretion of Phenolsulphonephthalein as a Test for Renal Function.— 
(Method of Rowntree and Geraghty.) 

Apparatus and Reagents Required. — (I) Hypodermic syringe, all 
glass, accurately graduated and calibrated, preferably one of Becton- 
Dickenson manufacture, the type referred to as "Tuberculin," with a 
long slender barrel. (2) Ampoules each containing 1 cc. of sterile 
phenolsulphonephthalein sterile solution, representing 6 mg. of the dye. 
These are marketed by Hynson, Westcott and Dunning, of Baltimore, 
Md. (3) Standard dilution made by diluting 6 mg. of the phenol- 
sulphonephthalein with a little water, adding 10 cc. of 25 per cent, 
sodium hydrate, and bringing the volume to 1000 cc. with distilled 
water. If not exposed to strong light this keeps for a period of months. 
(4) Solution of sodium hydrate (10 to 25 per cent, maybe used, usually 
15 per cent). (5) One thousand cc. graduate or volumetric flask. 
(6) Colorimeter. The colorimeter used for other colorimetric work 
may be employed, either the Duboscq, Kober, Bock-Benedict, or Myer. 
The Autenrieth-Koenigsberger colorimeter made by Hellige of Freiberg 
was modified for this work and has been very generally employed 
(Fig. 74). It has the advantage that the percentage may be read 



TESTS FOR DETERMINATION OF RENAL FUNCTION 



269 



directly from the scale. Hynson, Westcott and Dunning have put out 
a small inexpensive colorimeter, which is simple though not accurate. 
Procedure.— The patient is given a glass of water to drink to ensure 
free urinary secretion. One-half hour later the bladder is emptied, 
by catheterization if necessary. A sterile hypodermic syringe is filled 
with exactly 1 cc. of the phenolsulphonephthalein solution, which 
contains 0.006 gm. of the dye. This is injected preferably into the 
lumbar mascular tissue. The area selected should be free from edema. 
The bladder is emptied one hour after the injection of phenolsulphone- 
phthalein and again at the end of two hours either by voiding or by 




Fig. 74. — Hellige's colorimeter. (Simon.) 



catheterization. The two specimens so obtained are examined 
separately. Enough sodium hydrate solution is added to alkalinize, 
so that the color will develop fully, and the total volume in each speci- 
men is brought up to 1000 cc. with clear water. If the color be faint, 
it is well to bring the volume up to only 500 cc. The cup of the colori- 
meter is filled with some of the resulting mixture and the reading taken. 
When a Hellige colorimeter is employed, the percentage may be read 
off on the scale directly. If the urine were diluted to 500 cc. only, the 
reading should be divided by 2. 

When it is desired to employ the Duboscq or a similar colorimeter 



270 EXAMINATION OF URINE 

dilute a small quantity of the standard solution already described 
(6 mg. of phenolsulphonephthalein in 1 liter of water) with an equal 
quantity of distilled water. The left hand cup of the colorimeter 
should be half-filled with this, and then adjusted to a depth of 10 mm. 
The urine, alkalinized and diluted as directed, is placed in the right- 
hand cup, which is then moved up and down until the two sides of the 
visual field are the same, when the reading is taken. The calculation 

is made by this formula; -^- = per cent, phenolsulphonephthalein 

excreted. For example, with the standard set at 10, if the reading 

were 20, the percentage of dye excreted would be ~r = 25. 

The smn of the reading obtained from the two specimens represents 
the percentage of the dye excreted in two hours. 

Estimation of the Function of the Separate Kidneys.— To accomplish 
this purpose, catheterization of the ureters is necessary. The patient 
is given 200 to 400 cc. of water to drink about a half hour before the 
test to ensure the free flow of urine. For the technic of ureteral cathe- 
terization, the reader is referred to standard texts on genito-urinary 
surgery. After the catheters have been introduced, Rowntree and 
Geraghty direct that the cystoscope be withdrawn, placing around the 
free extremity of one of the catheters a bit of adhesive to distinguish the 
right from the left. It is well to be certain that there is a free flow of 
urine from the catheters before injecting the drug, in order to avoid the 
possibility of obscuring the results should there be inhibition of the 
urinary flow due to ureteral catheterization. In these cases, the drug is 
usually given intravenously rather than intramuscularly, on account 
of quicker elimination. As it is the relative amount of work each 
kidney is capable of doing that is being studied, it is sufficient to collect 
the urine from the ureter catheters for a period of twenty minutes 
after the appearance of the drug on one side. Provided neither ureter 
catheter becomes plugged during this twenty-minute collection period 
just as accurate a comparative estimate of the secretory power of the 
two sides can be made as if the ureter catheters were allowed to remain 
in position for a longer time. The ends of the catheters are placed in 
test-tubes so that the urine may be collected. Into each test-tube 
are placed a few drops of 15 per cent, sodium hydrate. The tubes are 
watched for the first appearance of the red color and the interval of 
time since injection is noted. 

Normal Results.— In normal cases the drug appears in the urine after 
intramuscular injection in from five to ten minutes and in from three to 
five minutes after intravenous administration . Forty to 60 per cent, of 
the amount given is recovered at the end of the first hour and from 15 
to 25 per cent, at the end of the second hour, making a total recovery 
for the two hours of 61 to 85 per cent. The excretion is markedly 
lower in all forms of nephritis, and is apparently more or less pro- 
portional to the amount of involvement of renal tissues. In chronic 



TESTS FOR DETERMINATION OF RENAL FUNCTION 271 

passive congestion, it is low, but returns rapidly to normal with 
improvement in circulation, while it fails to do so when the passive 
congestion is accompanied by nephritis. In surgical kidney conditions 
where the ureters can be catheterized and the amount of drug excreted 
by each kidney compared, the test often gives extremely valuable 
information. In a kidney which is the seat of hypernephroma, 
advanced tuberculosis, or calculus pyonephrosis, the time of appear- 
ance of the drug will be greatly delayed, and the amount may vary 
from a mere trace to a very small amount, while from the other side, a 
normal time of appearance and an amount of drug nearly equal to the 
excretory power of two kidneys will point to satisfactory compensation 
by the normal kidney. 

With urinary obstruction from hypertrophy of the prostate or 
stricture, and resulting back pressure on the kidneys, the time of 
appearance is delayed, and the amount of drug excretion diminished. 
With the relief of this back pressure, the recuperative power of the 
kidneys may be gauged by the degree of increase of elimination of the 
drug in a series of tests, over a period of days or weeks. The test is 
of value in diagnosing uremia from conditions which simulate it, and 
by very low readings, in indicating the fact that uremia was impending 
even when clinical evidence is absent. 

With this, as with all other functional tests, too much reliance 
cannot be placed on the findings in any single test or series of tests. 
Though strikingly accurate information is secured by its use in many 
cases, in others, the results do not check with other clinical findings 
and are difficult to explain. For this reason the data secured by the 
use of the test should be correlated with all other available clinical 
evidence before coming to a decision in any case. 

Excretion of Indigo-carmin.— The use of indigo-carmin in chromo- 
ureteroscopy has been advocated by Thomas. The onset, character, 
and intensity of coloration of the dye as it is ejected from the urethral 
orifices after intramuscular injection is observed cystoscopically. 
After introducing an evacuation-irrigation cystoscope, under local or 
general anesthesia, 20 cc. of 0.4 per cent, solution of indigo-carmin at 
blood heat are injected deep into the gluteal muscles. The bladder is 
drained, washed thoroughly, and filled with sterile boracic acid solution 
(150 cc. in the case of males, 200 cc. in females). Maintaining a 
brilliant illumination, the bladder wall is studied carefully. Not only 
the time of appearance of the dye but the intensity of the color should 
be noted. The appearance of jets of blue urine from the urethral 
orifices renders urethral catheterization easy in many instances where it 
might be extremely difficult to see the openings. From normal kidneys, 
indigo-carmin is eliminated as dark blue in from four and a half to 
twenty minutes; as light blue not later than fifteen minutes. Renal 
function is seriously impaired when the color does not appear in fifteen 
to twenty minutes. 

Thomas feels that this test is applicable in surgical affections of the 



272 EXAMINATION OF URINE 

kidneys and ureters, especially in hydronephrosis, pyonephrosis, pye- 
lonephritis, and chronic tuberculosis involving approximately one- 
third of the parenchyma; but is of no value in movable kidney, in the 
absence of urethral kink or hydronephrosis, pyelitis, early tuberculosis, 
most forms of nephritis, and small tumors not involving the greater 
part of the parenchyma. Thomas has described an "index of elimina- 
tion," determined by dividing the quantity eliminated during the first 
hour by the quantity eliminated during the third hour after injection. 
The index for a series of normal individuals was 5.1. He thinks that 
when the amount excreted during the third hour equals or exceeds that 
eliminated during the first hour, serious operative intervention, namely 
prostatectomy, is contra-indicated, unless the total amount eliminated 
in three hours exceeds 20 per cent. 

For the determination of quantity, the urine is collected by catheter- 
ization or voluntary micturition, and placed in 1000 cc. volumetric 
flasks, reserving a flask for each of three periods of one hour each. Clear 
water is added to each specimen to bring the total volume to 1000 cc, 
and portions are compared in the colorimeter with a standard prepared 
by placing 20 cc. of 0.4 per cent, solution of the dye in a volumetric 
flask and adding distilled water to 1000 cc. When used in the Hellige 
colorimeter, the percentage is read directly on the scale. For the 
Duboscq or similar instruments, a portion of the standard is diluted 
with three times its volume of distilled water and used in the left hand 
cup of the colorimeter which is set at 10 mm., while the diluted urine 
is placed in the right hand cup, comparing in the usual way, and calcu- 
lating with the following formula: 

~P^ = per cent, indigo-carmin excreted. 

Mosenthal Test-meal for Renal Function.— The patient is directed 
to take a full meal of an ordinary dietary, with the usual amount of 
meat, but to take neither fluid nor food between meals. The prohibi- 
tion against taking fluid between meals should be emphasized. During 
the day when the patient is taking this diet, the urine should be 
collected at two-hour intervals, the bladder being emptied each time as 
completely as possible. The specimens are kept in separate bottles, 
properly labelled with the time at which the contained urine was 
voided. % A time schedule should be given to the patient. For example, 
he should be directed to collect the specimens at 8 and 10 a.m., and 
12 n., and at 2, 4 and 6 P.M., and again three hours after the evening 
meal. Any urine passed after this time during the night should be 
kept and mixed with the specimen voided in the morning, which would 
represent the output of ten or twelve hours. 

Mosenthal states that under these circumstances the normal response 
would show a maximum specific gravity of 1.018 or more. The 
specific gravity should vary at least '.> points between the highest and 
lowest readings. The night urine should be small in amount, that is, 
less than 400 cc., and of high specific gravity, 1.018 or over. 



TESTS FOR DETERMINATION OF RENAL FUNCTION 



273 



Signs of a diminished renal function are, (1) a lowering of the maxi- 
mal specific gravity; (2), a fixation of the specific gravity; and (3) a 
nocturnal polyuria. 

Mosenthal has proposed a table giving various degrees of impair- 
ment as indicated by the results of the test-meal when compared with 
the results of other tests. 



Degree of impair- 
ment of renal 
function. 


Night urine. 


Variations in specific gravity 
when the highest specific gravity is 




Cc. 


Sp. gr. 


1.018 


1.017-15 


1.014-13 


1.012andless 


Normal . 
Slight . . . 
Moderate . 
Marked 
Maximal 


400- 
401-600 
601 - + 


1.018 + 
1.016-17 
1.015 — 


9 + 

8.5 
4- 


6 + 

5 and 4 

3- 


6 + 

4 and 5 

3- 


6 + 
5- 



The salt and nitrogen content of the various specimens may be 
determined also, but Mosenthal states (and most workers agree with 
him) that the simple procedures of measuring the volume of the urine 
and of determining the specific gravity yields sufficient data to give an 
adequate idea of the renal function in many respects. The Mosenthal 
diet test is easily employed and is of especial value in early cases of 
nephritis, for it gives positive indications and reaches the highest degree 
of impairment before other tests. 

It is to be borne in mind that so-called " dissociation of renal func- 
tion" is seen in some of the anemias. There is a marked fixation of 
the specific gravity at a low level and a nocturnal polyuria, while the 
amount of blood urea, the phenolsulphonephthalein test, and Ambard's 
coefficient show findings little difference from normal. 

Ambard's and McLean's Coefficients.— Ambard claims to have shown 
that the elimination of at least certain substances is carried on by the 
kidneys according to definite laws which are capable of mathematical 
expression. These laws he reduced to a mathematical formula known 
as Ambard's coefficient. The relative constancy of this as regards the 
excretion of urea was claimed by Ambard. 

The formula has been modified by McLean so that the index may 
express directly the changes in the rate of excretion. One hundred 
represents the normal finding and corresponds with a value of 0.08 
for Ambard's coefficient. In this country, the McLean index has 
come into wider use than the Ambard coefficient. 

McLean's index is expressed as follows: 



Index 



(gm. urea per twenty-four hrs.) V gm. urea per liter of urine X 8.96 
Wt. in kilos. X (Gm. urea per liter of blood) 2 



In order to secure free excretion of urine, the patient should drink 
150 to 200 cc. of water. Half an hour later the bladder is emptied 
completely, by catheter if necessary. The time is noted exactly to a 

18 



274 EXAMINATION OF URINE 

minute. The urine is collected at the end of seventy-two minutes. This 
time period is selected for the sake of convenience, since it is one- 
twentieth of twenty-four hours. Should an error of a few minutes 
be made of the second specimen in the collection of urine, the exact 
time should be noted and the output for twenty-four hours should be 
calculated on this basis. The amount of urea is determined in this 
specimen. 

Thirty-six minutes after the beginning of the period, 10 cc. of blood 
are obtained from the vein of the arm, clotting being prevented as 
usual by the addition of sodium or potassium oxalate (see page 167) 
and the amount of urea determined (see page 177). 

The patient must take neither food nor fluid during this period. If 
any urine be voided during the interval, it must be added to that 
obtained at the end of the period. The urine must be carefully col- 
lected and handled with care to avoid any loss, and the total quantity 
should be measured accurately. The patient's weight should be taken 
on the day of the test. 

The following data is now available : the quantity passed in seventy- 
two minutes, which may be multiplied by 20 to give the twenty-four- 
hour amount; the gm. of urea per liter of urine, from which may be 
determined the amount in twenty-four hours ; the grams of urea per liter 
of blood ; and the patient's weight in kilograms. 

These figures may be substituted in the formula. The calculation 
may be greatly simplified by a slide rule adapted to the formulas 
(Fig. 75). This may be secured from the Keuffel and Esser Co., 127 
Fulton Street, New York City. 

Example.— McLean gives the following example for calculation of 
the index by the aid of the slide rule : 

Gm. urea excreted per twenty-four hours, D = 20.0 

Gm. urea per liter of urine, C = 11.0 

Gm. urea per liter of blood, Ur = . 33 

Gm. body weight, in kilos, Wt = 55.0 

Index = D ^Tx 89 6 = 20.0 VlU) X 8.96 
Wt. X Ur 2 55.0 X (.33) 2 

This may be worked out with the slide rule as follows: 

1. 55.0 on the weight scale is set opposite 20.0 on the I) scale; first 
position. 

2. TJie hair-line on the runner is moved to 11.0 on the C scale; 
second position. 

3. The slide (center portion) is moved so that 3.3 on the Ur scale is 
at the hair line on the runner; third position. 

4. The reading is now made at the arrow which is pointing toward the 
scale /. In the example (see illustration), it points to 100, and the 
index is therefore 100. 

Interpretation of Results. McLean has shown that the findings 
correspond very closely with those of the phenolsulphonephthalein 



TESTS FOR DETERMINATION OF RENAL FUNCTION 275 

test. While 100 is taken as representing normal, it is stated that 
usually the index is between 100 and 200, and variations between SO 
and 300 are not infrequent in normal persons. He states that an index 




276 EXAMINATION OF URINE 

below 80 is to be considered as abnormal, though not necessarily 
seriously so. In renal disease an index below 50 is indicative of a con- 
siderable degree of renal impairment of functional ability. It is 
believed by him that the amount of damage to the kidneys is increas- 
ingly greater as the index is lower and tends to approach zero. A low 
index, however, may be only temporary, as in passive congestion of the 
kidneys due to heart failure or in acute nephritis, and may return to 
normal by improvement of the underlying condition. 

DETERMINATION OF TOLERANCE TO SODIUM BICARBONATE AS 
A TEST FOR ACID INTOXICATION. 

Method of Sellards. 1 — Sodium bicarbonate is given by mouth or 
intravenously in 5 gm. doses every two or three hours to determine the 
amount which is needed to make the urine neutral or alkaline to litmus 
(or to reduce the reaction to that of normal blood, pH 7.4). The 
bicarbonate should be given in a moderate amount of water and the 
patient should void before each administration. Specimens of urine 
which are not distinctly acid to litmus should be boiled. Boiling 
converts the bicarbonate to carbonate so that the reaction to litmus 
will be sharper. 

Normally 5 to 10 gm. are sufficient to change the reaction, while 
with acidosis more will be required, even as much as 140 gm. in severe 
cases. Palmer and Van Slyke have called attention to the possibility 
of giving unnecessarily large and possibly injurious amounts of bicar- 
bonate, since in most of the pathological cases which they studied the 
urine did not become neutral until the blood plasma bicarbonate content 
had reached a much higher figure than normal. They think that the 
therapeutic use of bicarbonate should be controlled by a determination 
of the plasma bicarbonate. They found that for each 42 pounds of 
body weight, it is necessary to give 0.5 gm. of sodium bicarbonate to 
raise the plasma C0 2 1 volume per cent. The amount given should be 
sufficient to raise the plasma bicarbonate 71 to 75 volume per cent. 

BACTERIOLOGICAL EXAMINATION OF URINE. 

For bacteriological examination, the urine should be obtained with 
great care. A catheter should be passed under aseptic conditions and 
the urine drawn into a sterile container. 

Examination of Urine for Tubercle Bacilli. — The urine should be 
obtained as described above, as though it were desired for cultural 
purposes. Fill 2 centrifuge tubes with urine and centrifuge for five 
minutes. Then pour off the supernatant fluid, add fresh urine, and 
again centrifugalize. Repeat the operation a number of times, until 
the sediment represents that obtained from 150 to 200 cc. of urine. 

1 Principles of Acidosis, Harvard University Press, Cambridge, 1910, p. 107. 



THE TWO- AND THREE-GLASS TESTS 277 

If a precipitate of urinary salts be present, the sediment should be 
"washed" with freshly distilled water by filling the tubes with distilled 
water and centrifugalizing for five minutes. The supernatant fluid is 
poured off and the process repeated. The sediment now may be 
spread upon a slide, adding a drop of Mayer's egg-albumen if necessary 
to facilitate adherence. After drying and fixing in the usual way, the 
film is stained by steaming with carbol-fuchsin for two minutes, as 
directed in the chapter on Sputum. In decolorizing, especial pains 
must be taken to rule out acid-fast organisms other than the tubercle 
bacillus, especially the smegma bacillus. The slide should be decolor- 
ized and simultaneously counterstained by pouring Pappenheim's 
rosolic acid decolorizing fluid over the film and draining it off five or 
six times. Inoculation of two guinea-pigs with the sediment obtained 
by centrifugalizing the urine is desirable. 

For assistance in the identification of other organisms, reference 
should be made to the chapter on Bacteriological Methods. Those for 
which search is made commonly include, in addition to the tubercle 
bacillus, the gonococcus, colon and typhoid bacilli, the pyogenic cocci, 
and the streptococcus. 

THE TWO- AND THREE-GLASS TESTS. 

Simple tests sometimes used for localizing the site of an inflammatory 
condition in the urethral tract are the two- or three-glass tests. In 
any of these tests the patient should have held his urine for at least 
four hours previous to making the tests. Conical glasses or tall 
cylinders with a capacity of about 200 cc. should be employed. 

Two-Glass Test.— The urine is voided into the first glass until about 
30 cc. are obtained when the flow is stopped. The remainder is 
allowed to pass into the second cylinder. The first glass is presumed to 
contain the washings from the urethra; the second glass will receive 
that from the organs which are proximal to (above) the external sphinc- 
ter, that is, the posterior urethra, prostate and bladder. However, 
the test may be misleading, since with posterior urethritis there may be 
little discharge from the posterior urethra to escape into the bladder so 
that the second specimen may be clear, giving rise to the false impres- 
sion that the anterior urethra is the seat of the trouble. Then with a 
simple anterior urethritis, there may be little urine to void so that the 
anterior urethra is imperfectly cleansed and the second glass is cloudy, 
when with a greater quantity of urine, it would be clear. 

Three-Glass Test.— This testis similar to that just described, except 
that the urine is passed in a continuous stream into three glasses, into 
the first two as it is in the two-glass test, and into the last with as much 
straining as possible, so as to express secretion from the prostatic 
ducts. It is subject to the same fallacies as the two-glass test. 

Four-Glass Test.— A more accurate estimate of the source of inflam- 
matory products in the lower urinary tract may be secured by the 



278 EXAMINATION OF URINE 

four-glass test. To carry this out, the patient must report with a 
moderately well rilled bladder. The test is done as follows: 

Glass I. Anterior Urethral Washings.— Before the patient voids any 

urine, the anterior urethra is gently flushed out with sterile water by 
means of a hand syringe or irrigator. These washings are collected in 
the first glass, and any pus or shreds will be obviously of anterior 
urethral origin. 

Glass -. Post-urethro-vesicaL— The patient now voids into the 
second glass what he considers to be a third of his bladder urine. Pus 
shred or blood contained in this glass must originate from the posterior 
urethra or bladder. 

Glass S. Vesical.— The patient voids into a third glass a further 
portion of his urine which may fairly be held to represent unmixed 
bladder urine. 

Glass 4- Prostato-vesicular. — The patient's prostate and vesicles 
are now massaged, after which he voids the remainder of his urine 
into a fourth glass. This glass should contain abnormal elements 
from the prostate and vesicles if they are diseased. 

Examination of Prostatic Fluid. — -In infection of the prostate or when 
there is a history of previous gonorrheal infection it is always 
desirable to examine the prostatic fluid. This is obtained by pros- 
tatic massage. The patient is directed to bend over, leaning against 
a table for support. The operator inserts the index finger, well greased, 
and covered with a rubber cot into the rectum and strokes the 
prostate from above downward with gentle massage first on one lobe 
and then on the other, until secretion appears. This is caught as 
it drops from the meatus upon a slide. In case no strippings are 
secured at the meatus, the urine should be voided immediately and 
the sediment examined. 

URINARY FINDINGS IN MORBID CONDITIONS. 

The diagnosis of renal diseases should not be made from the results 
of urinary examination alone. In a consideration of the clinical evi- 
dence, the urinary findings, no matter how complete, furnish only 
part of the evidence. Other features which should be given full con- 
sideration are the patient's history, the condition of the heart and the 
bloodvessels, the blood-pressure, the presence and absence of edema, 
and the presence of signs of uremia. The results of determinations of 
the amount of urea, creatinine, and uric acid and the CO2 combining 
pow T er of the plasma, furnish data of great value in the study of nephri- 
tis. The significance of the findings have been referred to in the 
section on Blood Chemistry. 

The classification of renal disorders which is used here is unsatis- 
factory both to pathologists and clinicians. It is utilized partly because 
it is followed in most of the standard works on medicine, but chiefly 
because no better one has been ottered for clinical purposes. 



URINARY FINDINGS IN MORBID CONDITIONS 270 

Acute Nephritis.— The quantity of urine is greatly reduced, even to a 
daily output of 100 to 150 cc. The color is dark, usually smoky, 
rarely a bright red. The specific gravity is high, usually over 1.025. 
The sediment shows blood corpuscles in various stages of hemolysis, 
epithelial cells, and casts. The casts include those of the hyaline and 
granular variety, and those containing red cells, epithelium, leukocytes. 
Albumin is present in large amounts. The phenolsulphonephthalein 
excretion is reduced. Should recovery ensue, there is a gradual im- 
provement in all these signs. The quantity of urine increases, the 
albumin diminishes in amount; the red cells disappear while casts 
often persist for a longer period of time. A definite proportion of cases 
which make a partial recovery gradually develop chronic nephritis, 
and the urinary findings are those which are expected in this condition. 

Chronic Parenchymatous Nephritis.— The specific gravity is usually 
increased in proportion to the diminution in the amount of urine. The 
color is yellow, rarely smoky. Albumin is present in rather large 
quantities. Microscopically, the sediment is seen to include occasional 
red cells, an increased number of leukocytes, hyaline, granular, epithe- 
lial casts, and occasionally fatty and red blood cell casts. There are 
also epithelial cells from the kidney and the pelvis. Phenolsulphone- 
phthalein excretion is impaired. The blood often does not show 
as great retention of nitrogenous substances as is seen in chronic 
interstitial nephritis, but may show a decided increase in the amount 
of chlorides. 

Chronic Interstitial Nephritis.— This includes the secondarily con- 
tracted kidney (small white kidney) and the primary granular kidney. 
From the standpoint of urinary findings, there is no way of differentia- 
ting between these types. Usually the urine is much increased in 
amount. Nocturnal urination may be the first symptoms of which the 
patient complains. The color is light yellow and the specific gravity 
is persistently low, ranging from 1.005 to 1.012. Albumin may be 
found in a small quantity but is often absent, and it may be necessary 
to examine a number of specimens to detect any. Casts, which are 
usually of the hyaline variety, with possibly a few of the granular type, 
are usually in a rather small number and may be absent at times. In the 
arteriosclerotic form of contracted kidney, the quantity is normal or 
reduced, the specific gravity is normal or high, there are hyaline and 
granular casts, and albumin is present, varying somewhat in amount 
according to the diet and exercise. The phenolphthalein excretion 
diminishes as the pathological process advances. Urine obtained after 
a Mosenthal test diet shows fixation of the specific gravity and an 
increase in the nocturnal quantity. The blood shows an increase in the 
amount of uric acid in many incipient cases; an increase in uric acid and 
urea in moderately advanced cases; and increases in these two sub- 
stances and creatinine as well in cases of a still more severe type. 

Amyloid Kidney.— In this rather uncommon condition the urine is 
increased in amount, pale and clear, of low specific gravity, and usually 



280 EXAMINATION OF URIXE 

shows an abundant amount of albumin. Hyaline, granular, and fatty 
casts, occasionally those of amyloid type, are found. 

Pyelitis. —The urine is turbid, due to the presence of pus. In addition 
to pus, there may be large amounts of mucus and desquamated epithe- 
lium. Blood is occasionally present, giving the urine a smoky appear- 
ance. Albumin is present in amounts ranging from a trace to a 
measurable quantity. The pus often varies in amount. It may be 
intermittent when only one kidney is involved and the ureter is tem- 
porarily blocked . At times, casts of the kidney tubules or moulds of the 
calyces may be passed. The reaction of the urine varies according to 
the tj-pe of the infecting organism. With a B. coli communis infection, 
it is usually highly acid. Microscopically, one finds leukocytes in 
various stages of preservation, depending upon the reaction. The 
outline is usually well preserved in acid urine, while in alkaline urine, 
the white cells are often partially dissolved, so much so that definite 
morphology may be almost distinguishable. In addition epithelial 
cells are seen, not infrequently those of the caudate type predominating. 
Red blood cells are seen at times. Since pyelitis is almost always 
accompanied by an involvement of the kidney substance itself, casts 
are to be expected. They may be hyaline, but it is not unusual to 
find them containing leukocytes, epithelial cells and red blood cells. 
Microorganisms are present, the varieties more frequently responsible 
for pyelitis including the tubercle bacillus, bacilli of the colon group, 
B. proteus vulgaris, and the staphylococci. 

Nephrolithiasis.— All cases of renal stone when complicated by 
infection are accompanied by varying degrees of pyelonephritis so that 
pus may be seen in the urine over a long period of time. Indeed, the 
diagnosis of pyelitis is not complete until .r-ray examination has been 
made to exclude the possibility of calculus. The urine, which in the 
absence of complicating pyelitis may have been quite clear, will often 
show at the time of colic red blood cells ranging in number from a very 
few detected only microscopically, to a quantity sufficient to render the 
urine turbid. 

Passive Congestion.— The kidneys are frequently involved in other 
conditions, to which reference has been made in discussing the causes 
of albuminuria and hematuria. The passive congestion occurring 
in failure of cardiac compensation is of especial importance from the 
standpoint of differential diagnosis. 

The amount of urine is small, at times almost to the point of anuria. 
It is dark in color, often smoky or of reddish tinge, due either to the high 
concentration or the admixture of blood. The specific gravity is high, 
and the reaction usually acid in freshly voided specimens. Albumin 
may be present in large amount. The sediment, which may be con- 
siderable, shows casts of the hyaline and granular varieties, not infre- 
quently those of epithelial and red blood cell types. Red blood cells 
and renal epithelial cells may be seen. The excretion of phenolsulphone- 
phthalein is low but increases as cardiac compensation is restored. 



URINALYSIS WITH RELATION TO LIFE INSURANCE 281 

Cystitis.— Urination is usually frequent and painful. The urine is 
turbid. Alkaline decomposition in freshly voided specimens is frequent 
in chronic cases, the sediment showing amorphous and triple phos- 
phates. In acute or chronic B. coli infections, the reaction is almost 
invariably acid. Pus may be present in macroscopic amounts. Mucus 
is seen at times, occasionally enough to form a ropy deposit. Albumin 
is present. Microscopically, red cells, epithelial cells (chiefly of the 
large round variety), leukocytes, and bacteria are found. Casts are 
not seen unless there be an involvement of the kidneys also. 

Urethritis.— The urine contains pus in varying quantities. Occa- 
sionally red blood cells may be seen in the acute stage, and albumin is 
often present when the inflammatory reaction is severe. The two-, 
three-, or four-glass test may be of assistance in localizing the exact seat 
of the process. 

Diabetes Insipidus.— The characteristic finding in this condition is a 
large quantity of pale urine of low specific gravity (1.001 to 1.005) 
containing no albumin or sugar. 

Diabetes Mellitus.— The amount of urine is large, ranging from 3 
even to 20 liters. It is generally of light color and of highly acid reac- 
tion. Albumin is not infrequently present. Sugar is present in the 
form of dextrose, the amount ranging from a fraction of a per cent, to 
10 per cent, (the latter figure representing a maximum which is seldom 
reached). The percentage is of course of secondary importance, the 
total amount per diem being of greater significance. This may range 
from 300 to 640 gm., and Osier states that there are exceptional cases 
with a daily output of from 1 to 2 pounds. In the more severe cases, 
acetone, diacetic acid, and /3-hydroxybutyric acid are present and the 
amount of ammonia is high. Chemical examination of the blood is of 
importance, and the laboratory procedures should include a determina- 
tion of the amount of blood sugar and of the C0 2 combining-power of 
the blood plasma. 



URINALYSIS WITH RELATION TO LIFE INSURANCE. 

To life insurance companies, the results of urine examinations assume 
great importance. The examiner should perform this part of his 
examination with the same care which he bestows upon the rest of the 
work. In taking the history, especial attention should be paid to the 
frequency of urination, nocturnal urination, and to pain on urination. 
The urine should be passed in the presence of the examiner. In the 
case of women, it should be passed in an adjoining room, and the 
examiner should be morally certain that it had been passed by the 
applicant. He can reassure himself to a certain extent by determining 
that the specimen is warm. Specimens should not be examined which 
are brought or sent in by the applicant. 



282 



EXAMINATION OF URINE 



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URINALYSIS WITH RELATION TO LIFE INSURANCE 283 

.The routine examination required by most companies includes the 
gross appearance, the determination of specific gravity, the reaction to 
litmus, the detection of albumin and sugar. The clarity or turbidity 
should be noted. The cause of turbidity should be explained on the 
blank, for the company will desire to know whether turbidity be due to 
the presence of bacteria or pus, or merely to a precipitation of urates 
or phosphates. Instructions for differentiating have been given in 
foregoing paragraphs (see page 218). The specific gravity is of 
importance chiefly to obviate the possibility of examining a dilute 
specimen, in which it is more difficult to detect a small quantity of 
albumin. The limits ordinarily set are between 1.010 and 1.130. The 
reaction is of slight importance except that alkalinity often indicates 
decomposition. 

Albumin should be tested for first by the heat and acetic acid test. 
Should this prove negative, the result may be taken as final, but if it be 
positive, Heller's test should be done. The result of both examina- 
tions should be noted, and should the examiner think that they indicate 
the presence of nucleoprotein, he should make a notation to this effect 
on the blank. The majority of the companies do not desire the use of 
more delicate tests than those mentioned, because the significance of 
faint traces found with these methods is not definitely determined. 
The companies generally prefer to have the examination for sugar 
carried out with one of the reduction tests. There is still some pref- 
erence for Fehling's solution. The solutions should be kept in separate 
bottles and should be mixed on the day when the test is made as directed 
in foregoing paragraphs (see page 223) . The urine should be fresh to 
prevent possible loss of sugar by decomposition. When a positive 
reduction is obtained, the presence of sugar cannot be taken as definitely 
established until the result has been checked by some method other than 
one employing the reduction principle. The fermentation test is 
simple and reliable. 

A determination of the amount of urea is sometimes required. It is 
practically impossible to obtain a twenty-four-hour specimen for 
insurance purposes, and it is difficult to see what useful purpose is 
served by ascertaining the amount of urea in a single specimen except 
to demonstrate' that the specimen is sufficiently concentrated to 
correspond to the specific gravity and that there is an approximate 
correspondence between specific gravity and urea concentration. The 
quantitative determination of other bodies, such as chlorides, phos- 
phates, sulphates, and uric acid, can have no possible significance when 
performed on a single specimen without regard to the diet. 

In making the report all abnormalities should be noted upon the 
blank, but they should not be reported to the home office until a 
second specimen has been obtained. Should the same abnormality be 
found again, the fact should be noted with the date of the second 
examination, and the blank sent to the company. If the second speci- 
men should prove normal, this fact should be noted and the blank 



2S4 EXAMINATION OF URINE 

should be sent to the home office, which usually requires that a third 
specimen be obtained and sent to them by mail for examination. "When 
either albumin or sugar has been found once, even though later speci- 
mens are clear, the applicant is required usually to sign a statement 
that his diet has not been restricted during the preceding six months 
for glycosuria or albuminuria (as the case may be), in order to protect 
the company against the possibility of accepting a poor risk who has 
been placed by medical care in favorable condition for examination. 

Microscopic examinations are made usually only at the home office 
of the company or by certain delegated referees or chemists at the 
larger centers. When sending a specimen for microscopic examina- 
tion, the examiner should take pains to prevent decomposition by 
prompt mailing and by the addition of a suitable preservative. Both 
container and preservative are usually furnished by the company. 

When the microscopic examination is to be performed away from 
the home office, it is desirable that the sediment from at least 15 cc. 
of urine be obtained by the use of a high speed centrifuge. As much of 
the sediment as possible should be placed upon the slide. One company 
has prescribed the following requirements which must be met before 
doubtful bodies are classed as casts: (1) Both ends should be rounded. 

(2) Neither end should show tapering, tendency to clubbing, or tails. 

(3) The sides should be parallel, and the body should show no cracks 
or indentations. (4) The matrix should be homogenous, except, of 
course, in the case of granular or cellular casts. (5) At least three 
perfect specimens should be found before the specimen is reported as 
positive. 

Opinions differ about the acceptability of applicants whose urine 
shows few hyaline casts, some companies accepting risks where the 
number is limited to a few in a given slide. This is done because it is 
recognized that hyaline casts do not always mean kidney disease, for 
they may result from indiscretions in diet or from excessive exercise. 
Granular casts, however, usually mean permanent kidney damage and 
almost invariably are taken as a cause for rejection. The attitude 
regarding the presence of casts has been summed up by a writer on 
this subject as follows: "The presence of a few hyaline casts should 
be treated in the same way as albuminuria. If they persist, the 
application should be postponed until the inspection of several speci- 
mens at some future time has demonstrated the fact that hyalines are 
no longer present. As long as they are present in the urine there must 
be some uncertainty as to whether the process through which they are 
formed is serious or inconsequential, and this opinion holds even 
though the condition of the applicant is otherwise satisfactory." As 
the same writer remarks, the life insurance point of view and that of 
the practitioner is quite different, since when a person applies for life 
insurance, the examination is necessarily limited to a short time and 
there is little opportunity for continued observation. 



CHAPTER III. 
EXAMINATION OF GASTRIC AND DUODENAL CONTENTS. 

GASTRIC CONTENTS. 

Relative Simplicity of Examination of Gastric Contents.— The examina- 
tion of gastric contents may be reduced by the practitioner to very 
simple terms. The more useful procedures can be carried out with a 
limited equipment and with the expenditure of a relatively small 
amount of time. 

It is highly important to select the proper meal to be given for the 
purpose in mind, to give more than one type of meal if the circum- 
stances demand, and, in many cases, to repeat the gastric analysis 
before drawing final conclusions. If, for example, the presence or 
absence of lactic acid might have considerable influence in making 
the diagnosis, the meal should be one which contains no lactic acid. 

Finally, the results must be interpreted in the light of the clinical 
findings, and should never be viewed as self-sufficient facts. As has 
been remarked by Dock, "the possibilities are that in stomach diag- 
nosis, as in other lines, the greatest assistance will come from the 
patient efforts to appreciate the alterations of function and structure 
primarily, and by allowing our diagnosis to grow out of these findings, 
assisted by the other clinical features of the case. . . . Examina- 
tions of gastric contents give information that cannot be obtained in 
any other way, and, though often negative, cannot be excluded without 
risk of serious error. Such examinations are indicated in all stomach 
syndromes, in all diseases seriously affecting metabolism and nutrition, 
and in all diseases that affect the function of the stomach. . . ." 

Apparatus required. — The apparatus required is included in the list 
to be found in the Appendix for "Minimum Equipment for the 
Clinical Laboratory." 

THE GASTRIC JUICE. 

General Considerations.— A brief review of gastric physiology is 
necessary in order to present the considerations underlying the examina- 
tion of stomach contents for diagnostic purposes. The first stimulus 
to gastric secretion is a psychic one, and depends upon the gratification 
of appetite and the pleasure in eating pleasing food. The stimulus 
caused by psychic influence suffices to cause an adequate flow of gastric 
juice for a limited period of time. This may last for only fifteen or 
twenty minutes. Further secretion is excited by the products of pro- 



286 EXAMINATION OF GASTRIC AND DUODENAL CONTEXTS 

tein digestion which act upon the mucous membrane of the stomach 
and produce a hormone, called gastrin, which when carried by the 
blood to the cells of the gastric glands, excites them to activity. 
Furthermore, it is known that fat has a distinctly inhibitory effect 
upon the secretion of gastric juice, and that the presence of oil in the 
stomach materially diminishes the amount of secretion poured out 
upon a meal which would be otherwise readily digested. The most 
abundant secretion is obtained with meat, while the digestive power is 
greatest after a bread meal. Therefore in evaluating the results 
secured after a test-meal, the type of meal must be given due con- 
sideration and the results which are regarded as standard for a stipu- 
lated meal would not be used as a criterion for an entirely different 
sort of meal. 

The amount of material recovered when the stomach tube is passed 
will depend to some extent upon the reaction of the contents, since we 
know that when digestion has reached a certain point and when the 
food has been churned into a homogeneous mass (chyme) of the 
proper reaction, the propulsion of portions of this mass against the 
pyloric sphincter will result in the opening of the sphincter. Contact 
of this acid material with the duodenal mucous membrane, however, 
causes the sphincter to close. Different kinds of food materials are 
discharged differently. The discharge of fats begins slowly and 
continues at a slow rate over a long period of time. The discharge of 
carbohydrates, on the other hand, begins soon after ingestion and 
rapidly becomes abundant, reaching a maximum in about two hours 
and usually leaving the stomach empty in three hours. The discharge 
of proteins is about intermediate between that of fats and that of 
carbohydrates, beginning in about half an hour and not attaining a 
maximum for four hours. The rates are dependent upon the combining 
power of the food material for acids, the carbohydrates having practi- 
cally no combining power and leaving the acid which is secreted free 
to open the sphincter, whereas fats inhibit gastric secretion. 

It has been shown that the effect of hyperacidity is to delay the 
emptying of the stomach. Even after the stomach has emptied itself 
of food, there is more or less continuous flow of gastric juice, as has 
been shown by Carlson, the amount varying from a few cc. to 60 cc. 
per hour. 

The functions of the hydrochloric- acid have to do with the control 
of the stomach movements, such as opening the sphincter, and with the 
secretion of the gastric juice and bile. Its chemical functions are 
concerned with the digestion of proteins by assisting pepsin; with the 
inversion of the dissaccharides; and with the antiseptic action, which 
prevents the growth of bacteria, notably yeast sarcinse, lactic acid and 
butyric acid bacilli, streptococci, etc. The action of pepsin and hydro- 
chloric acid upon the proteins is in the nature of hydrolysis. The 
first products are acid albumin, after which in succession conic primary 
proteose, secondary proteose, and peptone, While for many years 



TYPES OF TEST-MEALS 287 

the presence of another enzyme in addition to pepsin, i. e., rennin, 
has been assumed, this view has met with some skepticism of late and 
there is a strong feeling that the clotting of milk is due to pepsin. 

Characteristics.— The gastric juice is a thin, highly acid liquid, with 
a specific gravity ranging from 1.001 to 1.010. The amount of hydro- 
chloric acid varies from 0.1 to 0.3 per cent. The amount of pepsin 
is small by actual weight. Salts are present, largely chlorides and 
phosphates. A certain amount of gastric juice is present even in a 
fasting stomach. As seen in clinical work, the gastric juice is almost 
always mixed with partially digested food. 



TYPES OF TEST-MEALS. 

The normal findings vary for the different types of meals and this 
should be considered in evaluating the results. The meals should be 
given when fasting, preferably the first thing in the morning. Only 
such forms of food should be given as will not interfere with the action 
of the indicators which are to be employed. 

Ewald Meal.— This is the meal which is most frequently employed in 
clinical work, and has retained a degree of popularity in spite of many 
modifications which have been introduced. This is probably because 
long usage has taught the clinician just what results to expect in the 
average normal cases. The meal consists of a piece of white bread 
without crust, or a roll, to be taken with a cup of tea or a glass of water. 
The patient should be instructed specifically to take no butter on the 
bread and to use no milk or cream in the tea, and should be told to 
chew the bread thoroughly and to eat slowly. He should be directed 
to take no medicine before or after the meal. The meal should be 
removed about one hour after ingestion. Normally the amount 
recovered is between 20 and 60 cc. 

Riegel Meal.— While this meal or one similar to it may be used to 
determine the general digestive power, it is not particularly well 
adapted for the purposes of chemical investigation. The meal as 
ordinarily given consists of a cup of bouillon, 200 gin. of finely chopped 
cooked beef steak ("Salisbury steak"), two slices of white bread, 
150 gm. of finely mashed potatoes, and a glass of water. It is usually 
removed at the end of three or four hours. If the stomach be practi- 
cally empty at the end of three or four hours with a small amount of 
contents recoverable, the motility is regarded as normal, whereas if 
the stomach contain a considerable amount of food at the end of four 
hours, the digestive power is low. The amount of acid after such a 
meal is usually higher than would be obtained after an Ewald meal. 

Boas Meal.— This consists of 500 cc. of oatmeal soup, made by boiling 
a tablespoonful of oatmeal with 1000 cc. of water, reducing the bulk of 
the water by boiling down to a volume of 500 cc. The patient is 
instructed to use no cream, butter, or milk. This meal is removed in 



288 EXAMINATION OF GASTRIC AND DUODENAL CONTENTS 

from one to two hours, or even longer. It is given as a meal which 
contains no lactic acid. 

Dock Meal.— When it is necessary to give a meal free from lactic 
acid, Dock's meal would be given preference by most patients to Boas's 
on the grounds of palatability. It consists of a shredded wheat 
biscuit moistened with hot water and a cup of hot tea. 

Salzer Method of Determining Motility.— The patient is directed to 
take a meal consisting of one ounce of roast beef or broiled lamb chop, 
250 cc. of milk, 50 gm. of rice, and 1 soft boiled egg. Four hours later 
an Ewald meal is given, and one hour after the gastric contents are 
withdrawn. The presence of meat fibers is evidence of deficient pro- 
pulsive activity of the stomach wall. 

REMOVAL OF GASTRIC CONTENTS. 

The selection of the equipment which is used for the purpose of 
recovering the stomach contents is a matter of considerable importance. 
The tube should not be of too great caliber. The outside diameter 
should not exceed 12 mm. It should be provided with an opening 
at the end and with at least one at the side about an inch above the end. 
This is important since when one opening becomes occluded the con- 
tents may still be secured through the other. The side opening is not 
so likely to become occluded by contact with the gastric mucosa as 
the one on the end (Fig. 76). The writer prefers to use a Pollitzer 
bag for the purpose of securing the necessary suction (Fig. 76). It is 
easier to handle than more complicated outfits and there is nothing to 
get out of order. The tube is simply moistened with warm water 
before introducing it. Though the patient may assume either the 
recumbent or sitting posture; the former is preferable. After reassur- 
ing the patient a towel or rubber apron should be placed over his 
clothing while the operator should protect himself with an apron. The 
patient should take out any removable artificial teeth and should be 
directed to tip the head forward slightly. The mouth should be only 
partially opened. The Pollitzer bag is compressed and attached to 
the tube which is then introduced over the tongue, to one side of 
the epiglottis. The greatest gentleness should be used. The patient 
should take deep sighing breaths while the tube is in the esophagus. 
If the Pollitzer bag be compressed as directed until the esophagus has 
been entered, and if pressure be then released, the wall of the esophagus 
will grip the tube tightly until the tube enters the stomach, when air 
will rush into the bag, distending it. The length of the esophagus 
varies, so that the black ring which is used as a guide is of little assist- 
ance. The bulb and tube should be connected by a thick piece of glass 
tubing, so that it will be possible to note the passage of contents into 
the bag. 

When the contents do not appear after this procedure, certain steps 
may be taken to expedite their recovery. It is possible that the 



REMOVAL OF GASTRIC CONTENTS 



289 



orifices of the tube have been plugged with pieces of food material. 
The bag may be detached and allowed to fill with air, when it may be 
re -attached to the tube and air forced through very gently, withdrawing 
the tube a little at the same time. Then the bag is disconnected and 
re-attached for the purpose of making suction. If this maneuver is 
unsuccessful, the tube may be withdrawn for two or three inches. It is 
the writer's opinion that for the average person, the black mark should 




Fig. 76. — -Aaron's improved stomach tube and bulb. 



be placed farther down the tube, that is, the tube should not be intro- 
duced quite so far as the mark indicates. Gentle massage can be 
applied over the stomach and is always helpful. 

Contra-indications to the Use of the Stomach-tube.— There are certain 
rather generally accepted contra-indications to the use of the tube. 
It should not be passed in a case of aneurism, nor in one of uncom- 
pensated heart disease. Recent severe gastric hemorrhage, especially 
19 



290 EXAMINATION OF GASTRIC AND DUODENAL CONTENTS 

if from a gastric ulcer, or perforation of stomach, constitute contra- 
indications. 

Obtaining Gastric Contents with the Duodenal Tube and the Fractional 
Method of Examination.— An Einhorn or Rehfuss tube (Fig. 77) should 
be provided. There should he a glass syringe of 20 cc. capacity, which 
may be attached to the free end of the tube. 

The patient may be given a meal of the Ewald type, two slices of 
bread and ten ounces of tea or water. When this is employed, the crust 
should be removed from the bread, and especial precautions should 
be taken to see that mastication is thorough. At the close of the meal 
the tube is introduced. Two or three ounces of tea may be reserved 
and may be taken with the tube to facilitate its passage. The patient 
should be in the sitting posture. He is directed to tip the head back and 
the metal olivary body is slid into the pharynx over the dorsum of the 
tongue. Usually its further passage may be accomplished by directing 
the patient to swallow repeatedly. The tip usually reaches the lumen 
of the stomach before the 50 cm. mark on the tube passes the incisor 




Fig. 77. — Rehfuss tube, a, metal olivary body; b, rubber tubing; c, glass syringe. 



teeth. The contents are now obtained by aspirating with a syringe. 
A small quantity, i. c, a little over 5 cc, may be removed every fifteen 
minutes until the close of digestion. The tube may be secured in place 
by attaching to it one end of a strip of adhesive tape, fastening the 
other end of the adhesive tape to the face. Each specimen should be 
examined separately for the amount of free HC1 and total acidity. 
The small residues remaining may be pooled and utilized for the tests 
for lactic acid, blood, bile, and for microscopical examination. In 
making the titrations, it is desirable to use 1 cc. quantities of contents 
and ,-fj, sodium hydrate, multiplying the results by 20 to reduce to the 
usual terms (■/. e., number of cc. of -*, NaOH required to neutralize 
acid in 100 cc. of gastric contents). If desired, a cup of hot beef 
bouillon may be given instead of the Ewald meal. If bouillon be used , 
it should be remembered that the acidity curve is likely to be higher. 
Some workers prefer to introduce the tube when the bouillon is given, 
using the bouillon as a lubricant to assist in the passage of the tube. 

From the chemical determination made on the successive fractions 
of gastric secretion, a curve may be constructed in which the acidity 



ROUTINE EXAMINATION 291 

per cent, (the number of cc. of y^- NaOH required to neutralize acid in 
100 cc. of contents) is placed on the vertical line, and the time intervals 
are spaced along the horizontal base line. 

Rehfuss states that with this tube it is possible to determine the 
end-point in gastric digestion by failure to aspirate material where the 
patient is placed in different position; by the character of the last 
samples removed which are thicker and smaller in quantity; by the 
character of the sound produced by insufflation and heard by ausculta- 
tion over the stomach; and by lavage. He and his co-workers studied 
the curve in presumably normal individuals and found that there were 
three types of curves, which they term respectively isosecretory, 
hypersecretory, and hyposecretory. In the first there is a steady rise, 
a high point of total acidity of about 60 sustained for about half an 
hour, and then a gradual decline with total disappearance of food 
residues in from two to two and one-half hours. The hypersecretory 
type shows a rapid response with increase in acidity to a high point of 
70 or 100 or over, and a slow decline. In the hyposecretory type the 
curve is similar to the isosecretory with a slower response to stimuli, 
a slow ascent to the curve, and a high point of 40 to 50. They 
feel that the technic commonly employed for determining gastric 
function is entirely inadequate, inasmuch as it indicates but one phase 
in a constantly changing cycle, and because that phase is by no means 
always the high point in the digestive curve. 

ROUTINE EXAMINATION. 

The routine examination should include the following : (1) Note the 
type of meal and the time which elapsed between ingestion and removal. 
(2) Macroscopic characteristics, especially amount, color, odor, 
amount of food material; amount of mucus, if any; separation into 
layers ; presence of recognizable blood or bile. (3) Qualitative chemical 
tests, free HC1; blood and bile, if macroscopic appearance or patient's 
history arouse suspicion on either score; lactic acid. (4) Quantitative 
chemical tests, free HO; total acidity; combined acids, when free 
HC1 is absent. (5) Microscopic examination. 

Order of Procedure.— After noting the data about the type of meal and 
the time it remained in the stomach, the macroscopic appearance 
should be studied and recorded. The contents should be allowed to 
stand in a conical glass for at least fifteen minutes. Then a drop is 
placed on each of two or three slides for microscopic study. The 
major portion is filtered, preferably through gauze or filter paper while 
a small quantity, five or ten drops, is filtered through moistened filter 
paper and is used for the lactic acid test. While filtration is proceed- 
ing, the microscopic examination may be made. When this is com- 
pleted, the filtrate should be ready for the chemical tests. 

Gross Appearance.— The gross appearance often furnishes informa- 
tion of great importance, and its study should not be neglected. The 
color, separation into layers, and amount are of particular significance. 



292 EXAMINATION OF GASTRIC AND DUODENAL CONTENTS 

A mount. —If no food be found after an Ewald meal which has been 
removed at the end of one hour, this fact may be accepted as evidence 
of hypermotility. Normally, there is a return of from 20 to 60 cc. 
Large quantities (200 to 300 cc.) point to gastrosuccorrhea, motor 
insufficiency, or an obstruction at the pylorus. Motor insufficiency 
may be seen in anemia, pulmonary tuberculosis, and acute infectious 
diseases. Obstruction at the pylorus may be produced by carcinoma, 
syphilitic disease, or pressure from without the stomach, such as that 
produced by malignancy, adhesions, etc. 

Color.— After an Ewald meal the color of the fluid portion of the 
contents is white or grayish-white. Bright red blood may come from 
the nose or throat, or merely from an injury inflicted in the passage of 
the tube. When due to trauma, the amount is usually small. A gastric 
or esophageal ulce/r may be the source of blood. When the blood 
has been shed in the stomach, or has been swallowed, it is usually 
thoroughly mixed with the contents and is brown in color due to the 
action of the acid upon hemoglobin. When so mixed and changed in 
color identification should be completed by means of tests for occult 
blood. 

Occasionally the contents show a greenish-yellow color due to the 
presence of bile, which may have been forced back from the duodenum 
in retching. Gmelin's test for bile may be employed to confirm this 
finding. The presence of bile on several occasions indicates a stenosis 
of the duodenum below the opening of the common bile duct. A green 
color is not always due to bile, however. 

Odor. — Usually the gastric contents have a slightly sour odor in 
normal cases after the meals ordinarily given. A markedly sour odor 
is caused by butyric or acetic acid, or the presence of drugs, such as 
valerianic acid. The odor of putrefying flesh is sometimes noted in 
cases of gastric carcinoma where there is much necrotic tissue. A 
fecal odor may be detected where the intestinal contents have been 
forced back up into the stomach in intestinal obstruction. 

Amount of Food Material.— The relative proportion between food 
material and fluid should be noted. 

Presence of Mucus.— If mucus from the upper respiratory tract be 
swallowed, it may be seen in the contents and may be recognized by its 
dark color, due to the presence of inhaled carbon if the individual be a 
city dweller and by its stringy appearance when the stirring rod is 
drawn through the contents, while stomach mucus is clear, sago-like 
in appearance, and forms a homogeneous layer on top of the test-meal. 

Layers.— Usually the contents separate into two layers. When 
placed in a cylindrical graduate, it will be seen that in normal cases 
there is an approximately equal amount of granular food material 
below and comparatively clear fluid above. In cases of hypersecretion, 
the fluid portion is relatively greatly increased, while with hyposecre- 
tion, the consistency is thick since there is little fluid. In cases of 
pyloric stenosis and catarrhal gastritis, there is usually a considerable 



ROUTINE EXAMINATION 293 

quantity of contents of which on standing separates into three layers, 
the upper one being mucus, the middle clear fluid, and the bottom food 
particles. In severe catarrhal processes, the abundant mucus may 
carry up food particles into the upper layer. 

Qualitative Chemical Tests.— Free Hydrochloric Acid. — This may be 
tested for very simply with either Congo-red or with dimethyl-amino- 
azobenzol. The first indicator will give a blue color in the presence of 
free hydrochloric aeid, the latter a red. In using either indicator, a 
cubic centimeter or so of the stomach contents to be examined may be 
placed in a test-tube or evaporating dish and to it may be added a drop 
or two of the indicator in solution. 

Dimethyl-amino-azobenzol solution (Topfer's reagent) may be pre- 
pared by dissolving 0.5 gm. of the dry indicator in 100 cc. of 95 per cent, 
alcohol. Congo red is made by dissolving 0.5 gm. in 90 cc. of water and 
then adding 10 cc. of 95 per cent, alcohol. 

It is sometimes convenient to employ these indicators in paper form. 
Papers may be prepared by dipping filter-paper in a solution of the 
indicator. The paper is allowed to dry and is cut into small pieces, 
which may be preserved in bottles for a considerable period of time. In 
employing papers for a test, a drop of the stomach contents is touched 
to the paper when the characteristic color change should appear in the 
presence of free hydrochloric acid. 

Congo red gives a violet color with organic acids. 

Gilnzberg's reagent is preferred by some since it gives no color change 
whatever with organic acids. The reaction is striking and unmistak- 
able. The disadvantages are, that the solution does not keep indefi- 
nitely, and that the performance of the test requires a little more time. 
The procedure is, to dry two or three drops of the reagent in a clean 
porcelain evaporating dish or porcelain spoon very slowly and carefully 
over a low flame, taking especial care to prevent charring. With a 
glass rod a small portion of the stomach contents is touched to the film 
formed by the dried reagent. The dish is again warmed gently when 
a purplish-red color will appear if free hydrochloric acid be present. 
Giinzberg's reagent is prepared by dissolving 2 gm. of phloroglucin and 
1 gm. of vanillin in 100 cc. of 95 per cent, alcohol. 

Boas' reagent is preferred by many because of its greater stability. 
The test is performed in the same way. The reagent is prepared by 
dissolving 5 gm. of resorcinol and 3 gm. of sucrose (cane-sugar) in 100 
cc. of 50 per cent, alcohol. 

Lactic Acid.— The test for lactic acid which should be given prefer- 
ence in the clinical laboratory is Kelling's. Two test-tubes should be 
chosen which are of about the same bore. One is filled with distilled 
water and in it are placed two or three drops of 5 per cent, aqueous 
solution of ferric chloride. The color should be a very faint but 
unmistakable yellow. After thorough mixing the solution is divided 
equally between the two tubes. One tube serves as a control on the 
color . To the other are added two or three drops of the stomach contents, 



294 EXAMINATION OF GASTRIC AXD DUODENAL CONTEXTS 



25c.c. 



previously filtered through filter paper. When lactic acid is present, 

a yellow color appears. This is more readily apparent if the tubes be 

held over a sheet of white paper and viewed from the top in order to 

look through the depth of the column of fluid. 

It is preferable to free the stomach contents from substances other 

than lactic acid which may give the test and from substances which 
may interfere with the tests. A portion 
of the contents should be shaken up with 
ether. The mixture is allowed to stand 
for a few moments to permit its separa- 
tion into layers. The ethereal portion 
is pipetted off. This is evaporated to 
dryness, the residue is dissolved in water, 
and the solution used in making the test. 
A more convenient method for prepar- 
ing an ethereal extract is with the aid of 
a Strauss separatory funnel (Fig. 78). It 
is provided with two marks, one for 5 cc. 
and one for 25 cc. The stomach contents 
are poured in to the 5 cc. mark and ether 
is added to the 25 cc. mark. The upper 
end is stoppered and the mixture is shaken 
vigorously for a few moments. After this 
it is allowed to stand long enough for the 
ether to separate, when the stop-cock is 
opened and the contents are allowed to 
run out until the upper level of the fluid 
reaches the 5 cc. mark, that is, until there 
are only 5 cc. of ethereal extract left in 
the funnel. Water is added to the 25 cc. 
mark. This aqueous dilution may be 
employed for performing Kelling's test as 
described above. 

A rough quantitative test, sufficiently 
accurate for all clinical purposes, may be 
carried out by adding two drops of 10 
per cent, ferric chloride to the diluted 
ethereal extract in the Strauss funnel, 
shaking gently. It is stated (Wood) that 
with 0.1 per cent, of lactic acid, there will 

be a very intense yellow green color, while 0.05 per cent, will show 

only a slight green color. 

Uffelmann's test is not recommended for the detection of lactic acid 

in stomach contents, because of the interference caused by organic 

substances. 

Significance.— There are always small amounts of lactic acid present 

after meals which contain bread, milk, or meat. When the presence or 




5c.c 



Fig. 78.— Strauss' funnel for 
making lactic acid test. (Aaron.) 



ROUTINE EXAMINATION 295 

absence of lactic acid in the contents will have weight in the diagnosis, 
a lactic-free meal should be given. The presence of lactic acid after 
such a meal usually accompanies an anacidity and is due to the fer- 
mentation which takes place in the absence of hydrochloric acid. It 
may be found in gastric carcinoma but also in other conditions where 
retention and fermentation are seen, such as benign pyloric stricture, 
dilatation of the stomach, and permanent anacidity. 

Blood.— Blood may be recognized microscopically when it has not 
been digested by the action of acid. Usually, however, blood which has 
been in the stomach any period of time is altered and chemical tests 
are necessary for identification. The benzidine test may be used. In 
a test-tube are put 2 cc. of unfiltered gastric contents with 1 cc. of 
glacial acetic acid. To the mixture are added 3 cc. of a saturated 
alcoholic solution of benzidine. The contents of the tube are thor- 
oughly mixed and 5 or 6 cc. of fresh hydrogen peroxide are added. 
In the presence of blood a deep Prussian-blue color appears. 

Significance.— Blood may be seen in the stomach contents as a result 
of trauma inflicted in the passage of the tube. In such cases, it is 
usually unchanged in color, bright red, and often comes in streaks with 
a little mucus. It may be mixed with the stomach contents. In the 
latter case it may have been swallowed and have had its origin in the 
upper portion of the respiratory tract, in the mouth, or in the esophagus. 
Lesions of the stomach which may produce hemorrhage are, notably, 
gastric ulcer or gastric carcinoma. 

Bile.— The bile tests may be carried out as directed for urine, 
employing a filtrate of the contents. (See page 233.) 

Significance. — Bile in the stomach contents may be due to retching 
during the process of removing the test-meal. A green color in the 
stomach contents is not always due to the presence of bile, but may 
result from the presence of organisms containing chlorophyl. 

Quantitative Chemical Tests.— The quantitative determinations 
usually made on the gastric contents include a determination of the 
amount of free hydrochloric acid, of the total acidity, and at times of 
the combined acids. The acids in the stomach include free hydro- 
chloric acid, combined hydrochloric acid, organic acids, and acid salts. 
In the presence of free hydrochloric acid, the amount of organic acids 
is small. By combined hydrochloric acid is meant the hydrochloric 
acid which has combined with food-protein to form acid-albumin. 

It is of assistance to remember the end-points of the indicators and 
their significance. 

Dimethyl-amino-azobenzol, end-point, canary yellow; when reached 
means that the free mineral acids have been neutralized. 

Phenolphthalein, end-point, deep red; means that the free acids, 
combined acids, and acid salts have been neutralized. 

Alizarin, end-point deep violet; means that the free acid and salts 
have been neutralized. 



296 EXAMINATION OF GASTRIC AXD DUODENAL CONTENTS 

Procedure. — Filtered contents should be used. First one of the 
qualitative tests should be employed to determine whether free hydro- 
chloric acid be present or not. If absent, there should be determined 
only the total acidity, the combined acids, and the hydrochloric acid 
deficit. 

When free hydrochloric acid is present, the following procedure 
should be followed: A burette (Fig. 54,/) graduated in 0.1 cc. should 
be charged with decinormal sodium hydrate. The sodium hydrate 
should be run out until the nozzle is filled and the initial reading (A) 
taken and recorded. In a porcelain evaporating dish are placed 5 cc. 
of the filtrate to which are added about 25 cc. of distilled water and 
three drops of dimethyl-amino-azobenzol solution. A bright red color 
should appear in the presence of free hydrochloric acid, yrr NaOH is 
now added to the stomach contents drop by drop until the color becomes 
ochre-yellow, or until the last trace of pink just disappears, stirring the 
mixture in the evaporating dish with a glass rod. The free hydro- 
chloric acid has been neutralized when this color has been reached. 1 

To the same portion of partially neutralized contents, now ochre- 
yellow in color, 3 drops of phenolphthalein solution (1 gm. of phenol- 
phthalein dissolved in 100 cc. of 95 per cent, alcohol), should be added. 
The reading (C) of the burette is taken and the decinormal sodium 
hydrate is added while constantly stirring the mixture in the evaporat- 
ing dish until the color becomes a deep pink, in fact until the maximum 
color change has been secured. It is not sufficient to obtain merely 
the first permanent trace of pink, since if this color were taken as the 
end-point the acid salts would not be neutralized. When the proper 
end-point has been secured, the reading should be taken (D). Deter- 
minations should be made on a second portion of 5 cc, and the average 
taken. 

Calculation.— The results are ordinarily expressed in terms of the 
number of cc. of decinormal sodium hydrate which are required to 
neutralize 100 cc. of the gastric contents. The results obtained in the 
titration with 5 cc. of the gastric contents are therefore multiplied by 20. 

Example.— The original reading in the burette (A) was 5.5. The 
reading when the end-point for dimethyl-amino-azobenzol was reached 
(B) was 7.5. The reading when the titration with phenolphthalein 
was reached (C) was 7.5. The reading when the end-point for phenol- 
phthalein was reached (C) was 9.5. 

1 In many texts it is directed to carry the neutralization until a canary-yellow color 
appears. This method has been criticized by A. L. Benedict who states that while 
the color change from cherry-red to canary-yellow is almost instantaneous in titrating 
solutions of inorganic acids, the change in the stomach contents is very gradual, due to 
the presence of organic acids and to the organic combination of part of the hydro- 
chloric acid. He states that the reading for free hydrochloric acid should be taken 
at the point when the cherry-red tint disappears and is replaced by an orange tint, 
the Benedict's point is well taken and may be verified by adding to a second specimen of 
contents the amount of decinormal sodium hydrate ascertained by a titration made in 
the manner he prescribes. When a portion of this neutralized mixture is tested with 
Gunzberg's reagent, it will be found to contain no free HC1. There may be a difference 
of from 10 to 15 degrees in the final end-point as determined by the methods. 



ROUTINE EXAMINATION 297 

B — A =2. This multiplied by 20 gives 40, the number of cc. of deci- 
normal NaOH required to neutralize the free HC1 in 100 cc. of gastric 
contents. 

(B ,- A) + (D -C) =4. This multiplied by 20 gives 80, the number of 
cc. of decinormal NaOH required to neutralize total acids in 100 cc. 
of gastric contents. 

The number of cc. of decinormal sodium hydrate required to neutra- 
lize a given amount of stomach contents when multiplied by the factor 
0.00365 will give the actual amount of free hydrochloric acid in that 
amount of stomach contents, expressed in grams. 

Combined Acids.— When desired, the amount of the combined acids 
may be determined by titrating 5 cc. of filtered gastric contents with 
■J-Q NaOH, using a few drops of 1 per cent, aqueous solution alizarin 
as an indicator. The end-point has been reached when a deep violet 
has been obtained. One should become familiar with the end-point 
by adding a drop of alizarin to a 1 per cent, solution of sodium carbon- 
ate. Subtract this reading obtained from the reading obtained with 
the phenolphthalein as an indicator, multiply the remainder by 20, 
and the result indicates the combined acids in 100 cc. of gastric con- 
tents. 

Hydrochloric Acid Deficit.— When free hydrochloric acid is absent 
the "HO deficit" should be determined. Place 5 cc. filtered gastric 
contents in an evaporating dish and add 3 drops of dimethyl-amino- 
azobenzol solution. Add from burette -^0 HO until rose-red color is 
obtained. Take original reading from final reading, and multiply by 
20 to express the number of cc. of ^ HO required to neutralize 100 cc. 
of gastric contents. 

Discussion of Method,— The method which has been given is that of 
Topfer. While it is open to criticism, especially because dimethyl- 
amino-azobenzol reacts to organic acid in large quantity as well as to 
free mineral acid, and while the results may be not extremely accurate, 
it serves clinical purposes excellently. It is not satisfactory in the 
absence of free hydrochloric acid. The use of more elaborate and time- 
consuming methods is scarcely justified, particularly when it is remem- 
bered that the results after all test-meals are subject to variations. 
As has been remarked by Benedict (Medical News, 1904, lxxxxiv, 
597), "it really makes little difference which of several indicators is 
used provided one understands that the results are approximate and 
reliable only for comparative purposes. With low hydrochloric acid 
and high fermentation acidity, the readings of the burette are very 
inaccurate though they indicate the principles of therapeutics clearly 
enough." Whenever it is necessary to have an accurate test made, the 
chyme must be incinerated. 

Normal Results.— The free hydrochloric acid in a specimen removed 
one hour after an Ewald meal is usually between 30 and 40 (cc. of y^- 
NaOH required to neutralize free HO in 100 cc. gastric contents), 
representing about one-third to one-half of the total acidity. The 



29S EXAMINATION OF GASTRIC AND DUODENAL CONTENTS 

combined acid may be added to the free hydrochloric acid to ascertain 
the amount of useful acid secreted, since combined acid is acid which 
has partially fulfilled its function of digestion. The figures for com- 
bined acid vary greatly, averaging about 10 cc. There is, however, 
no rigid standard which can be set down as a normal for any of the 
findings, since marked variations are seen even in health. The results 
obtained by the fractional method show T that the maximum excretion of 
acid is reached in some cases before the end of the hour, after an hour in 
others. To set down the results obtained from a single determination 
as representing the patient's response to a meal, is open to obvious 
error. The figures vary with age, social condition, race and with 
nervous factors which may influence psychic or "appetite" secretion. 
The term eucJdorhydria is used to describe the presence of hydrochloric 
acid in normal proportions. The absence of hydrochloric acid is 
usually spoken of as anacidity of achlorhydria. Achylia is the condition 
in which neither hydrochloric acid nor enzymes are present. Sub- 
aridity or hypochlorhydria refers to a diminished secretion, and hyper- 
chlorhydria or hyperacidity to an increased secretion of hydrochloric 
acid. Achlorhydria is seen in advanced gastritis, in the neuroses, in 
gastric carcinoma and in achylia gastrica, which is commonly found in 
pernicious anemia. Hyperchlorhydria may be occasionally found in 
presumed health. It is also found in the neuroses, in beginning 
gastritis, and in ulcer of the stomach. Hypochlorhydria is seen in sub- 
acute or chronic gastritis, occasionally in the neuroses, at times in 
gastric or duodenal ulcer, in early carcinoma of the stomach, in the 
course of acute general infections, and in advanced tuberculosis. 

Microscopic Examination.— Microscopic examination is of com- 
paratively limited usefulness but should be carried out as part of the 
routine examination, since through it occasional clues are obtained. 
A drop of the unfiltered contents should be placed on a slide and 
covered with a cover-slip. The field should be darkened by dropping 
the Abbe condenser and by partially closing the substage diaphragm, 
since it is impossible to study the formed elements with a well-illumin- 
ated field. The examination should be conducted at first with the low 
power and later with the high power dry lens. The presence or 
absence of the following elements should be noted: elastic tissue, 
muscle fibers, fat drops, fatty acid crystals, starch granules, and organ- 
isms of various types, especially large bacilli, sarcinse, and yeast cells 
(Fig. 79). If testing with Lugol's solution or Sudan III be desired, a 
drop of the reagent may be placed on the slide next to one side of the 
cover-slip when a bit of filter paper may be touched to the fluid under the 
cover-slip at the other side, starting a current which draws the reagent 
under the cover-slip. 

Fat drops appear as globules of varying size of moderate refractility. 
They stain red with Sudan III. Fatty acid crystals are long, needle- 
like crystals which melt into globules. Sarcince are readily recognized 
by the typical bundle-like arrangement of the cocci. Yeast cells may 



ROUTINE EXAMINATION 



299 



he distinguished by the ovoid outline and the budding which some of 
the cells show. The only bacilli of special note are the Boas-Oppler 







Fig. 79. — Collective view of vomited matter. (Eye-piece III, objective 8 A, Reichert.) 
a, muscle fibers; b, white blood corpuscles; c, c', squamous epithelium; c", columnar 
epithelium; d, starch grains, mostly changed by the action of the digestive juices; e, 
fat globules;/, sarcinse ventriculi; g, yeast fungi; h, forms resembling the comma bacillus 
found by the author once in the vomit of intestinal obstruction; i, various microorgan- 
isms, such as bacilli and micrococci; k, fat needles, between them connective tissue 
derived from the food; I, vegetable cells, (v. Jaksch.) 



bacilli, which are found in large numbers when free hydrochloric acid is 
absent. They are long and exceedingly large rods, occurring in long, 
jointed chains (Fig. 80). With Lugol's solution, the Boas-Oppler 



V 1 <yt 



fS'iKiX 



Fig 80. — Boas-Oppler bacillus. (Simon.) 



bacilli stain brown, while mouth organisms of similar morphology 
(Bacillus maximus buccalis, Leptothrix buccalis) stain blue. The 



300 EXAMINATION OF GASTRIC AND DUODENAL CONTENTS 

Boas-Oppler bacilli are Grain-positive. The work of Gait and lies 
{Jour, of Path, and Bacter., 1914-1915, xix, 239) makes it appear 
that the Boas-Oppler bacillus is identical with B. bulgaricus, and that 
it is not an organism sui generis. It grows in the absence of hydro- 
chloric acid, and produces lactic acid. 



SPECIAL CHEMICAL TESTS. 

Pepsin.— The determination of pepsin is not usually required, since 
ordinarily pepsin is present when there is free hydrochloric acid. When 
free HC1 is absent, however, it is advisable to make tests for pepsin. 
The qualitative test, which is readily performed, serves all practical 
purposes. 

Qualitative Test for Pepsin.— Small discs of coagulated albumin are 
prepared from the white of an egg which has been coagulated by boiling 
in a test-tube placed in a water-bath. Boiling must not be allowed to 
continue beyond the point of coagulation, since it may render the 
albumin difficult of digestion. The block of egg-white is sliced into 
thin layers about 1.5 mm. thick with a safety-razor blade and small 
discs about 10 mm. in diameter are cut out of these layers with a sharp 
cork borer. The discs should be preserved in glycerol. One of them 
should be tested with normal stomach contents to ascertain the time 
which is required for complete digestion and the time so determined 
should be noted on the bottle. To perform the test, one of the discs 
is freed from glycerol by thorough washing with water and placed in 
about 10 cc. of stomach contents. If the contents contain no free 
hydrochloric acid they should be acidified with enough decinormal 
hydrochloric acid to turn Congo-red paper blue. The tube containing 
the contents and disc is then placed in a warm place, preferably in the 
incubator at 37° C, and shaken at intervals. At the end of two or 
three hours the disc should have dissolved. The exact time should 
be compared with that determined for the particular set of discs which 
were employed. 

Mett's Quantitative Method.— Mett's method gives approximately 
accurate results. Egg-white is diluted with an equal volume of water 
to precipitate the globulin, which is removed by filtration, using the 
suction pump if necessary. The filtrate is placed in a large test-tube 
or narrow beaker and in it are immersed a bundle of fine capillary 
tubes, about 2 mm. inside diameter and of convenient length. The 
tubes are allowed to stand completely submerged in the albumin solu- 
tion until free from air-bubbles, when the vessel with its contents is 
placed in a bath of boiling water for from five to fifteen minutes, depend- 
ing on the amount of fluid. Boiling should not be continued longer, 
since the coagulated egg albumen softens with prolonged heating. The 
tubes are removed from the vessel, the albumin on the outside is scraped 
oft' and the ends sealed with a little sealing wax. When used for tests, 



SPECIAL CHEMICAL TESTS 301 

sections about 2 cm. long are cut off and the sealing wax removed from 
the ends. 

The stomach contents are diluted 16 times with twentieth-normal 
hydrochloric acid (adding 15 cc. of ft HC1 to 1 cc. of the gastric con- 
tents). One of the Mett tubes is put in the diluted gastric contents 
and kept at 37° C. for twenty-four hours when the length of the 
column of albumin which has been digested is measured with a milli- 
meter scale, using a hand lens. In normal gastric juice, the upper 
limit of digestion is usually 4 mm. The amount of enzyme present is 
proportional to the square of the number of millimeters digested. For 
example, if one specimen digests 2 mm. and another specimen digests 
3 mm., the ratio between the enzyme values based on the squares of 
these figures, would be as 4 is to 9. 

Quantitative Determination of Pepsin (W. C. Rose's Method) 
(Arch. Int. Med., 1910, v, 459.)— Solutions Required— 1. 0.25 gm. of 
pea-globulin dissolved in 100 cc. of 10 per cent, sodium chloride solution 
(using gentle heat if necessary to secure solution), and the solution is 
filtered. 

Pea-globulin is secured by grinding about 2 handsful of ordinary 
garden peas (Pisum satirum), and by extracting them with 200 cc. of 
10 per cent, sodium chloride solution. The peas should be freed as 
much as possible from their outer coating when they are extracted 
repeatedly with large quantities of 10 per cent, sodium chloride. The 
extracts are combined and filtered through bolting cloth. After 
standing overnight in large cylinders to permit the deposition of 
insoluble material, the supernatant fluid is siphoned off, saturated with 
ammonium sulphate and filtered. The filtrate is discarded and the 
precipitate, containing the albumins and globulins, is placed in a 
dialyzer and dialyzed in running water for three days to remove the 
salt and dissolve the albumins. The globulins, which remain as a 
precipitate, are filtered, washed in water 2 or 3 times to remove all 
albumin, and dissolved in 10 per cent, sodium chloride. The resulting 
solution is filtered until perfectly clear and neutralized to litmus by 
adding dilute sodium hydroxide very carefully. Dialyzation is again 
employed for three days to remove the salt; the precipitated globulins 
are filtered and dried in a water-bath at 40° C. The proteins should 
be preserved during the entire process of separation by the addition 
of an alcoholic solution of thymol and toluene. Globulins prepared in 
this way dissolve almost completely in 10 per cent, sodium chloride, 
and after acidification with hydrochloric acid, yield a turbid solution. 

II. Hydrochloric acid (0.6 per cent.). This may be prepared from 
concentrated (36.5 per cent.) HC1 by adding 2 cc. of the latter to 118 
cc. of water. 

Procedure.— A known amount of the gastric contents is made neutral 
to litmus by adding ft NaOH. Should a precipitate appear, it is 
removed by filtration. Sufficient water is then added to dilute to 
five times the original volume. The diluted and neutralized stomach 
contents are then divided into two portions, one of which is boiled. 



:h)l ) examination of gastric and duodenal contents 

In each of 6 test-tubes place 1 cc. of the globulin solution and 1 cc. 
of the hydrochloric acid solution. Allow five minutes to elapse for the 
development of turbidity. With a 1 cc. pipette (graduated in 0.1 cc.) 
place in the tubes unboiled diluted gastric contents as follows: First 
tube, none; second tube, 0.1 cc; third tube, 0.3 cc; fourth tube, 0.5 cc; 
fifth tube, 0.8 cc; sixth tube, 1.0 cc. Then add enough boiled diluted 
gastric juice to bring the contents to 3 cc The table illustrates the 
method of setting up the tubes. 

Tube No 1 2 3 4 5 6 

Globulin solution, cc 1.0 1.0 1.0 1.0 1.0 1.0 

HC1 solution, cc 1.0 1.0 1.0 1.0 1.0 1.0 

UnboileddUutedgastricjuice.ee. . 0.0 0.1 0.3 0.5 0.8 1.0 

Boiled diluted gastric juice, cc. . . 1.0 0.9 0.7 0.5 0.2 0.0 

Incubate at 36° C. for one hour. Select the tube containing the 
smallest amount of gastric juice which shows no turbidity. This 
contains the least amount which completely digests the globulin. 

Results.— The enzyme content is expressed by the number of cc 
globulin solution that would be digested by 1 cc. of undiluted gastric 
juice. For example, tubes No. 1, No. 2, and No. 3 are cloudy, but No. 
4 is clear. This means that digestion of 1 .0 cc. of the globulin solution 
has been effected by 0.5 cc. of a 1:5 dilution of gastric contents, or by 
0.1 cc. of undiluted gastric juice. If 0.1 cc. would digest 10 cc. the 
enzyme content is expressed as 10 units. The normal results are about 
10 units. In cancer, the results range from to 3. The significance 
of enzyme determinations will be discussed under the consideration of 
the findings in various morbid condition. 

Quantitative Determination of Dissolved Albumin. — (Method of Wolf 
and Junghaus). 

Solu t ion Requi red : 

Phosphotungstic acid (pure) 3 cc. 

Hydrochloric acid (concentrated) 10 " 

Alcohol (95 per cent.) 200 " 

Aquoe dest., to make 2000 " 

This is kept in a bottle with a rubber stopper. 

Procedure.— Prepare a 1 : 5 dilution, (A), by adding 4 cc of distilled 
water to 1 cc. of gastric juice and a 1 : 50 dilution, (B), by adding 9 cc. 
of distilled water to 1 cc. of gastric juice. Place 1 cc. of distilled water 
in each of 6 test-tubes. To the first tube add 1 cc. of dilution (A), 
mix well by drawing a portion of the fluid into the pipette, ejecting 
back into the tube, repeating 2 or 3 times, and finally place 1 cc. of the 
mixture in the second tube. Mix as before, carrying 1 cc. of this 
mixture to tube 3, mix again, and discard 1 cc. 

Next prepare tube 4 by adding 1 cc. of 1 : 50 dilution (B), mixing the 
contents as before, and placing 1 cc. in tube 5. Repeat the operations 
until tube (I is mixed, when 1 cc. should be discarded. The series of 
tubes now contain 1 cc. respectively of 1 : 10, 1 : 20, 1 : 40, 1 : 100 
] : 200, 1 : 400 dilutions. To each add 1 cc. of the phosphotungstic 



SPECIAL CHEMICAL TESTS 303 

acid solution, layering it over the diluted stomach contents. A pearly 
white ring at the junction of the gastric juice with the reagent denotes 
a positive reaction. 

The secretion must be examined immediately after its withdrawal. 
The test-meal should be given on an empty stomach in order to guard 
against the presence of residues and the patient instructed not to 
swallow salivary or bronchial secretions while the meal is in the 
stomach. The test is applicable only to gastric contents which con- 
tain no free hydrochloric acid or blood. 

Results. — Normally positive reactions are obtained in dilutions 
from 1 : 10 to 1 : 50 in normal individuals. When present in dilutions 
of 1 : 100, 1 : 200, and 1 : 400, malignancy is indicated, according to the 
authors of the test. Friedenwald and Kieffer obtained positive results 
in 13.5 per cent, of 67 cases of benign achylias, and 83.9 per cent, 
positive or suspicious results in 106 undoubted cases of gastric cancer. 
Smithies found a positive result in 86 per cent, of 83 cases of gastric 
cancer, in 10 per cent, of 10 cases of pernicious anemia, in 17 per cent, 
of 17 cases of achylia gastrica. 

Volatile Fatty Acids. — The fatty acids found in the stomach contents 
are usually butyric or acetic acid. Butyric acid may be recognized 
by the pentrating odor, which is like that of rancid butter. The odor 
of acetic acid, similar to that of vinegar, is not so readily recognized. 

A test which may be employed for the volatile fatty acids is to place 
a few cc. of the contents to be tested in a test-tube and suspend a strip 
of moistened blue litmus in the mouth of the tube. The contents are 
heated. The volatilized acids will redden the litmus paper. 

For more positive identification, 15 or 20 cc. of the contents are 
shaken with about 2 gm. of sodium sulphate and 50 cc. of ether. The 
ethereal extract is separated and evaporated to small bulk preferably 
before a fan at room temperature (with care to avoid explosion) . The 
residue is divided into two portions which are treated as follows : 

One portion is dissolved in a few drops of water and a bit of calcium 
chloride added. Butyric acid will separate, since it is insoluble in 
calcium chloride, floating to the top as small fat drops, which will have 
the characteristic odor of rancid butter. 

The second portion is dissolved in a small quantity of water and 
neutralized with a 2 per cent, solution of sodium carbonate. A few 
drops of 5 per cent, ferric chloride solution are added, when a blood-red 
color appears if acetic acid be present. Upon boiling, basic ferric 
acetate separates out as a brownish-red precipitate. 

Significance.— Volatile fatty acids are not produced in the stomach 
under normal conditions, but may occur when there is stagnation 
and absence of free hydrochloric acid permits fermentation. 

Mercury.— Gastric contents may be examined for mercury by the 
method of Lee and Vogel (see page 266) . 

Fragments of Tumor Tissue.— Such bits of suspicious material should 
be fixed properly, sectioned and suitably stained. Occasionally, the 



304 EXAMINATION OF GASTRIC AND DUODENAL CONTENTS 

findings of carcinomatous material has furnished the final evidence 
in making the diagnosis of gastric carcinoma, but as a rule the pieces 
are too small for satisfactory examination. 

Absorptive Power.— The absorptive power of the stomach may be 
determined by giving the patient a few grains of sodium or potassium 
iodide in a gelatin capsule. After filling, the outside of the capsule 
should be carefully cleaned to remove the salt. The iodide will be 
excreted in the saliva promptly in normal individuals. Specimens of 
saliva, obtained at intervals of two or three minutes, should be treated 
with a few drops of starch solution and a few drops of nitric acid. A 
dark-blue color denotes the appearance of iodide. In normal indivi- 
duals, this is seen in from five to ten minutes. The test is not of great 
practical value, since occasionally the absorption is rapid even with 
chronic gastritis or dilatation of the stomach. 

SAHLI'S DESMOID TEST. 

Sahli has devised a method for testing the digestive power of the 
stomach, which is referred to as the desmoid reaction. In the center 
of a piece of rubber dam is placed 0.05 gm. of methylene blue. The 
dam is wrapped to form a small sack or bag, taking pains to keep the 
methylene blue inside the pouch, and is tied securely with No. 00 raw 
catgut which has been rendered pliable by prolonged soaking in cold 
water. The excess of rubber above the catgut is cut off. The capsule 
is washed carefully and tested in water to determine that there is no 
leakage of methylene blue. 

The capsule is swallowed at the close of a full meal. Beginning two 
hours later the urine is examined for green color at thirty-minute 
intervals. When the characteristic color appears in six to twenty 
hours, the gastric juice contains sufficient hydrochloric acid and pepsin 
to dissolve the catgut and liberate the methylene blue. Sahli claims 
that the pancreatic juice is incapable of digesting the catgut. 

FINDINGS IN VARIOUS STOMACH DISEASES. 

Gastric Ulcer.— The diagnosis rests upon a careful physical examina- 
tion and painstaking study of the symptoms with the confirmatory 
evidence furnished by the x-ray and the laboratory. Usually there is 
an aetive secretion of gastric juice of high acid content. This finding, 
however, is not by any means pathognomonic. Also indicative of 
ulcer is the presence of blood, especially if it be altered, and further 
evidence is afforded by finding blood in the stool after the patient has 
been placed upon a suitable diet (see page 314). It must be noted that 
all of these findings ma}' be given by a carcinomatous ulcer in its early 
stage. 

Dilatation of the Stomach (Gastrectasis).— The findings on physical 
examination of the lower border of the stomach far below the usual 



FINDINGS IN VARIOUS STOMACH DISEASES 305 

Level and of a succussion sound long after the stomach should have 
emptied itself, are of prime importance. Epigastric bulging may be 
seen. Frequently there is a history of vomiting large quantities of sour- 
smelling food. Usually a large quantity of contents is obtained after 
a test-meal, and unless stomach lavage be performed before the test- 
meal, residue of food taken during the preceding twenty-four or even 
forty-eight hours may be found. The acidity is variable, a low acidity 
being found in some cases, an hyperacidity in others. When the 
amount of free hydrochloric acid is low, lactic acid and the other 
organic acids may be found with many bacteria, especially sarcinse 
and yeasts. The amount of urine is usually low and the chlorides are 
often diminished. 

Carcinoma of the Stomach. —The history and physical findings are of 
primary importance and the results of the test-meal examinations must 
be regarded only as part of the evidence. Delayed motility is to be 
expected when the tumor is situated at the pylorus. Free hydrochloric 
acid is absent and the total acidity is diminished in many cases. These 
findings are by no means constant, however, since continued observa- 
tion has shown a normal amount of acid in the early stages in many 
cases. The presence of lactic acid depends, of course, upon the lack 
of free hydrochloric acid, as does the presence of the Boas-Oppler 
bacilli. Neither of the latter findings however, can be regarded as 
belonging exclusively to the picture of gastric carcinoma. Pepsin is 
diminished or absent. The amount of albumin in the contents may be 
increased. With ulceration, blood may be found in the contents and 
demonstrated in the stools. Occasionally fragments of tumor tissue 
are found in the water returned after lavage. Unfortunately in the 
early stage of a carcinoma, particularly of one which has developed 
upon an ulcer as a base, the test-meal shows nothing which may be 
regarded as characteristic. 

Achylia Gastrica.— While rare, this condition is of importance, espe- 
cially from a diagnostic standpoint. While seen most often in pernicious 
anemia, there is considerable possibility of confusing it with carcinoma 
of the stomach in differential diagnosis. The food returned in the 
test-meal shows no evidence of digestion. Free hydrochloric acid is 
absent, the total acidity is low, and the ferments are diminished. 
Lactic acid may be present, though only in small amounts. Often 
motility is normal. Repeated examinations of the contents should 
be made before the diagnosis is regarded as established. 

Continuous Hypersecretion (Gastrosuccorrhea, Reichmann's Disease). 
— In addition to the objective symptoms, repeated analyses of gastric 
content are of value. After lavage at night, there is found in the 
fasting stomach on the following morning a considerable quantity of 
gastric juice of high acid content containing no food remnants, yeasts, 
or sarcinte. The hydrochloric acid content is greater than normal with 
relation to the total acidity. After test -meals, the free hydrochloric 
acid content is increased and starch digestion diminished, Before 
20 



306 EXAMINATION OF GASTRIC AND DUODENAL CONTENTS 

making a diagnosis, such findings should be obtained on a number of 
occasions, excluding conditions which may cause a mere hyperchlor- 
hydria and demonstrating the secretion of a considerable quantity of 
highly acid digestive juice without food stimulus. 

Chronic Gastric Catarrh.— The amount of contents removed 'after an 
Ewald meal is normal or increased. After the condition has existed 
for a long time, there is a diminished total acidity and the free hydro- 
chloric acid is low in amount or absent. The food material shows little 
evidence of digestion. Considerable mucus is present in many cases, 
often enough to render filtration impossible. Pepsin is present, though 
usually diminished in amount. Starch digestion ordinarily proceeds 
well so that there will be little erythrodextrin and much achrodextrin. 
The motor function usually is not greatly impaired. 

The Gastric Neuroses.— Since this term is used to describe a number of 
vague symptom complexes, it is difficult to describe the gastric find- 
ings with exactitude. Before making a diagnosis of gastric neurosis, 
organic diseases must be excluded beyond question of doubt. In a 
given case of functional neurosis, the findings are often variable, since 
at times an anacidity, and at times an hyperacidity, is found. Indeed 
gastro-enterologists hold that only one type of case should be included 
under this classification, that is, one where gastric analyses show a 
more or less constant variation in gastric secretion, motility and 
sensation. While the acid findings are variable, the ferments are 
usually constant. Mucus is seldom seen in any quantity. 



EXAMINATION OF DUODENAL CONTENTS. 

Since the introduction of the duodenal tube by Einhorn it has been 
possible to secure the duodenal contents for examination. The 
practical value of examinations is not entirely settled, since there has 
not been a sufficient accumulation of data to permit competent judg- 
ment. 

Obtaining the Duodenal Contents.— A Rehfuss or an Einhorn tube 
may be employed. Robinson has encountered difficulty with the 
Rehfuss tube, which is of relatively large size with slits on the side, 
because it occasionally was plugged by the walls of the pylorus during 
its passage so that secretion could not be obtained, and recommends 
making a small hole in the end of the metal olivary body. He gives 
the following explicit description of the method which he used for 
introducing the tube : " The tube was passed as a rule on a fifteen-hour 
fasting stomach. The tip was introduced into the mouth when the 
patient was lying down. The patient then swallowed, and as soon as 
the tip passed into the esophagus he assumed the sitting posture, being 
requested not to swallow any more and to expectorate all saliva. By 
supporting the tube and having the patient breathe slowly and deeply, 
the tip slowly passes down the esophagus into the stomach. The tubes 



EXAMINATION OF DUODENAL CONTENTS 307 

are marked at 35 cm., 50 cm., 60 cm., 70 cm., etc., from the tip. When 
the 35 cm. mark passes the teeth, the tip is at the cardiac orifice. The 
patients were then placed on their right side. The tip then, together 
with what secretions are present, gravitates toward the pylorus. No 
secretions reach the pylorus. The distance from the pylorus to the 
mouth is indicated by the second mark (50 cm.) on the tube. Speci- 
mens were then obtained every ten minutes until the tip passed through 
the pylorus into the duodenum. This took, in normal cases, about 
thirty minutes." 

To determine whether or not the tube really is within the lumen 
of the duodenum, Einhorn gives the following points : If upon aspira- 
tion, the fluid is obtained quickly, looks watery, and shows free hydro- 
chloric acid, then one may know that the tube is in the stomach. If 
on the contrary, only a little material of neutral or alkaline reaction 
and of yellow color be obtained, it is duodenal fluid. When gastric 
anacidity is present, some confusion may arise, but when the fluid 
comes very slowly, the chances are that it is duodenal fluid. To settle 
the question, the patient may be given some fluid to drink which he has 
not had recently, such as milk colored with raspberry syrup. If the 
fluid obtained by immediate aspiration be clear and contains no milk, 
it is duodenal fluid, while if colored milk be obtained, the tip of the tube 
is in the stomach and not in the duodenum. The z-ray may be used 
to determine the exact location of the tube. 

Normal Appearance of Duodenal Contents.— Normally, the duodenal 
contents are clear, amber-yellow in color, and neutral or alkaline in 
reaction. Ordinarily about 15 cc. may be aspirated at first and then 
about 10 cc. every ten or fifteen minutes. Free hydrochloric acid is 
not present normally. Upon standing for several days the amber color 
will change to green, due to the oxidation of bilirubin to biliverdin. 
Microscopically only a few leukocytes, a few epithelial cells, and a few 
bacteria are seen in normal cases. 

Specimens should be examined macroscopically for gross character- 
istics, including color and the presence of mucus. Chemically they 
should be examined for free hydrochloric acid, total acidity, lactic acid, 
blood, bile, and possibly for ferments. Microscopically, search should 
be made for red blood cells, leukocytes, epithelium, bacteria, and gall- 
stone detritus. The technic for these examinations has been given in 
the section on stomach contents (q. v.) except for the tests for ferments 
which follow. 

Tests for Ferments.— The tests for trypsin are not especially satis- 
factory. Myers and Fine give the following as fairly satisfactory tests 
for amylase and lipase: 

Amylase.— Six test-tubes are placed in a rack. Into all but the 
first is placed 1 cc. of distilled water. Into both the first and 
second tubes is put 1 cc. of duodenal juice with a 1 cc. Mohr pipette 
(graduated in 0.1 cc). The duodenal contents and water in the 
second tube are mixed thoroughly by drawing them into the pipette 



308 EXAMINATION OF GASTRIC AND DUODENAL CONTENTS 

and ^ejecting a number of times, when 1 cc. of the mixture is placed 
in the third tube. The contents of this should be mixed thoroughly 
as before and 1 cc. put in the fourth tube. In the fifth tube, the 
process should be repeated, and 1 cc. of the mixture discarded. The 
sixth tube contains no duodenal juice and serves as a control. The 
tubes now contain respectively 1 cc, 0.5 cc, 0.25 cc, 0.12 cc, and 0.00 
cc. of duodenal contents. All six tubes receive 5 cc. of a 1 per cent, 
solution of soluble starch and are placed in the incubator at 38° C. for 
thirty minutes, when they are immediately filled with cold water. A 
few drops of y^ iodine (or Lugol's solution) are added and the tubes 
shaken. The tube is selected which shows a complete disappearance 
of all blue color, indicating complete conversion of the starch to achro- 
odextrin. 

Enzyme activity is expressed in terms of the number of cc of starch 
solution which 1 cc of duodenal contents is capable of digesting. If, 
for example, digestion is complete in the third tube (the one containing 
0.25 cc of contents) but not in the fourth tube, the activity is expressed 
as 20, since it is apparent that if 0.25 cc. digests 5 cc, 1 cc. could digest 
four times as much, or 20 cc. The activity varies between 5 and 200, 
the average being 40. 

Jjpasc.~T\vo test-tubes are taken. Into each are placed 1 cc. of the 
duodenal contents. One of the tubes is boiled. Heating serves to destroy 
ferment activity. Both tubes receive 1 cc. of neutral ethylbutyrate, 
10 cc. of distilled water and 1 cc. of toluene, and are shaken and placed 
in the incubator at 38° C. They should be shaken several times and 
at the end of twenty-four hours, the contents are transferred to two 
evaporating dishes. The acidity is determined by titration with #J T 
sodium hydrate, using phenolphthalein as an indicator and continuing 
the addition of the alkali until the first trace of permanent pink. The 
titration result obtained with the contents of the boiled tube (control) 
is subtracted from that obtained with the contents of the other tube 
and the resulting figure taken as the measure of the lipolytic activity. 
Myers and Fine state that they have obtained figures varying from 
0.3 to 4.3 cc, and the average has been 1.5 to 2.0 cc. 

Findings in Morbid Conditions.— A cloudy alkaline duodenal secretion 
is significant of an inflammatory condition in the gall-bladder, bile 
ducts, duodenum or stomach. The cloud is due to bacteria and pus. 
It is to be remembered that if hydrochloric acid be present in the 
duodenal contents, there will be turbidity due to the precipitation of 
the bile salts, and that this turbidity disappears upon neutralization. 
The duodenal secretion shows in some cases of cholecystitis where 
there are numerous small stones an orange-yellow, coarse, putty-like 
detritus. McNeil states that in one case of chronic pancreatitis, there 
was a constant diminution of pancreatic ferments and an absence or 
only a trace of lipase; three cases of achylia pancreatica showed con- 
stant absence of the pancreatic ferments; and in so-called duodenitis, 
the contents showed considerable stringy mucus, numerous Gram- 



EXAMINATION OF DUODENAL CONTENTS 309 

negative, motile bacilli, and cocci. He thought that the tube did not 
give any great amount of assistance in cases of duodenal ulcer. 

Bacteriological Study of the Duodenal Contents.— The duodenal tube 
has been suggested as a means of obtaining duodenal contents for 
bacteriological examination, particularly for the detection of typhoid 
carriers. MacNeal and Chace and Hess suggested a technic for 
securing specimens for bacteriological examination. They recom- 
mended the use of a perforated tip which has been sterilized in water, 
and over which had been slipped by means of sterile forceps a gelatin 
capsule, soaked in 95 per cent, alcohol for several days. The tube fitted 
with the capsule was dipped in shellac several times until well coated. 
Hess recommended blowing off the capsule when it is in place in the 
duodenum, introducing air for this purpose with a syringe. Lyon 
has the patient employ an antiseptic mouth wash, but does not try to 
cover the capsule during its passage through the stomach. 

Securing Bile by the Duodenal Tube.— Meltzer observed that the local 
application of a 25 per cent, solution of magnesium sulphate to the 
mucosa of the duodenum causes a complete local relaxation of the 
intestinal wall. Since the salt does not produce this effect when given 
by the mouth, that is, when it passes through the stomach before reach- 
ing the duodenum, he suggested its introduction into the duodenum by 
means of the duodenal tube, with the idea of causing a relaxation of the 
sphincter and the ejection of bile in cases of biliary colic and jaundice. 
He stated that 25 cc. of the solution would do no harm in adults while 
the dose in babies should not exceed 4 cc. 

Lyon has adapted this method to the study of diseases of the gall- 
bladder and biliary ducts. The procedure which he outlined is as 
follows : The patient is examined on a fasting stomach. The mouth is 
rinsed with an antiseptic solution, for example, potassium permangan- 
ate, 1 grain to 2 ounces of water. A sterile duodenal tube, fitted with 
a metal tip, is introduced into the stomach and some of the fasting 
residuum is aspirated and placed into a sterile vessel (Specimen 1). 
It is examined for consistency, the presence of mucus, and free and 
total acidity. 

When the tube has passed the pylorus, a syringe full of air is intro- 
duced into the duodenum. After the contents have been obtained and 
placed in a sterile bottle, 50 to 100 cc. of a 25 per cent, solution of 
magnesium sulphate are forced by the syringe into the duodenum or 
are allowed to flow by gravity and in a few minutes, gentle aspiration 
is started. The yellow color due to bile is usually seen in from two to 
ten minutes, becoming more pronounced as aspiration proceeds. When 
it deepens to a decided yellow, the material is placed in a sterile bottle 
and set aside (Specimen 2) . Lyon believes that the first bile aspirated 
is that present in the bile-ducts, while the darker, more viscid, and 
more concentrated bile is from the gall-bladder. He now starts 
collecting another specimen (Specimen 3), which is also placed in a 
sterile bottle, followed by thinner and more transparent bile of lighter 



310 EXAMINATION OF GASTRIC AND DUODENAL CONTENTS 

yellow color, which is aspirated much more slowly and intermittently. 
He assumes that this comes directly from the liver. 

Lyon asserts that in this manner it is possible to obtain bile sepa- 
rately from different portions of the biliary tract, and to study each 
specimen cytologically, chemically, and bacteriologically. He has found 
the bile from the gall-bladder to vary in amount from 30 to 100 cc. 
The largest amount in his experience was 166 cc. 

BILE IN PATHOLOGICAL CONDITIONS. 

Lyon has found that the bile in various pathological conditions 
showed the following variations from normal. 

Choledochitis.— The first bile collected was definitely different from 
normal in that it was more viscid and turbid, and contained an excess 
of flaky mucus. Cytologically, it showed pus-cells enmeshed in 
mucus, epithelial cells, and occasionally, red blood cells. Pathogenic 
organisms may be obtained on culture. 

Cholecystitis.— In cholecystitis without choledochitis, the first bile 
collected is relatively normal, macroscopically and cytologically, but 
the second specimen is markedly pathological, being more viscid than 
bile from the normal bladder. It is turbid with flaky or stringy 
mucus, and cytologically shows pus cells, occasionally red blood cells 
and desquamated epithelium. Cultures show pathogenic organisms. 
Lyon recovered streptococci of various strains, staphylococci, B. 
typhosis, B. pyocyaneus, and Micrococcus catarrhalis. 

Cholelithiasis.— With gall-stones, there is evidence of cholecystitis, 
but in a few cases there is in addition a gritty or sand-like sediment. 
In one case three small faceted stones were secured. 

Obstruction of the ducts would alter the picture in each instance. 



CHAPTER IV. 
THE EXAMINATION OF THE FECES. 

Introductory.— The examination of feces for clinical purposes requires 
only the simplest equipment and a small expenditure of time. Macro- 
scopic examination alone often reveals information of great importance. 
All the procedures given as steps in the Routine Procedure may be 
reduced to very simple terms with practice and should be employed 
as readily as the clinician resorts to urinalysis or gastric analysis to aid 
him in the solution of his problems. Indeed Stitt asserts that in the 
tropics, the examination of the feces is of greater importance than the 
examination of the urine. The unpleasant features attendant upon 
the examination of the feces may be reduced to a minimum by observ- 
ing the precautions which will be spoken of subsequently. 

Apparatus Required.— Except for the cultural methods, the apparatus 
required is included in the list to be found in the Appendix for " Mini- 
mum Equipment for the Clinical Laboratory." 

Composition of the Feces.— The feces are composed of undigested food, 
unabsorbed gastric, hepatic, pancreatic, and intestinal secretions, 
desquamated epithelium from the gastro-intestinal tract, debris of all 
sorts both from food material and from cells, and a large bulk of 
bacteria. It has been stated that the fecal excretion of an adult male 
will range from 110 to 170 gm. per day, and that the solid contents 
varies from 25 to 45 gm. With a coarse vegetable diet, the amounts 
both of total excretion and solid contents will be much greater. Bac- 
teria make up a large proportion of fecal material, possibly one-third 
of the total by weight. 



THE ROUTINE EXAMINATION. 

The routine examination should include the following : 

Macroscopic Examination.— Form, and consistency; odor; mucus; 
concretions; parasites; undigested food; pus; blood. 

Chemical Examination.— Reaction; presence of blood (when indi- 
cated, provided patient has been on an appropriate diet) ; presence 
of bile pigments (when indicated by history, and always when stool is 
of pasty color) . 

Microscopic Examination.— Food remnants; neutral fats and salts 
of fatty acid; red blood cells; leukocytes; parasitic ova; mucous parti- 
cles; general character of bacteria. 



312 THE EXAMIXATIOX OF THE FECES 



METHOD OF TRANSPORTING SPECIMEN. 

When a patient is directed to bring a specimen of feces to the labora- 
tory, it is advisable to furnish him with a glass jar, about 4 inches high 
and about 4 inches in diameter, fitted with a screw top. A Mason jar 
of one pint capacity makes an excellent container, or a jelly glass with 
tight fitting cover may be used. He should be told that the jar is to 
be only partially filled. In hospital practice, specimens may be sent 
to the laboratory in small white enamel or granite pails provided with 
covers. When a bottle or container is opened in the laboratory, care 
should be taken in removing the top, especially when the container is 
almost filled with feces, since the specimen often undergoes fermenta- 
tion and there may be a sudden and forcible expulsion of ill-smelling 
material. Care should be taken to have the specimen kept cold 
except when it is to be examined for amebpe, and delivered to the 
laboratory as promptly as possible, and to examine it at the earliest 
possible moment, before fermentation takes place. This is of especial 
importance when one desires to determine the extent of intestinal 
digestion, because under suitable conditions changes may take place 
in meat-fibers and other food remnants. 



MACROSCOPIC EXAMINATION. 

Macroscopic examination is of great importance. It should include 
a survey of the form and consistency, odor, color, the presence or 
absence of mucus, blood, pus, and undigested food. A search for 
parasites and stones should be made if clinical indications point to its 
necessity. 

Form and Consistency.— Any deviations from the well-recognized 
normal should be noted. Both form and consistency are to a great 
extent dependent upon the diet, so that even under normal conditions 
the consistency may vary from that of a thin paste to a solid stool. 
A watery discharge is, of course, abnormal. Extremely soft stools 
are seen after the use of purgatives and cathartics; serous stools are 
seen in Asiatic cholera; hard, knotty stools (scybala), are found in 
constipation; and flattened, ribbon-like stools, while occasionally seen 
in normal people, are also seen when there is a tumor, stricture or other 
partial obstruction low in the intestinal tract. 

Color.— The color of normal adult stool is dark brown. This is due 
to the presence of hydrobilirubin, which is formed from the bilirubin of 
the bile by the reducing action of certain bacteria. It is thought at 
the present time that hydrobilirubin and urobilin are identical. The 
character of the diet, however, has an important influence on the color. 
In infants, it is yellow, partly due to the milk diet and partly to the 
presence of bilirubin. With adults, an exclusive meat diet causes the 
feces to be dark, whereas after a milk diet they are light and yellow in 
color. Chocolate and cocoa give a dark color. Certain vegetables, 



MACROSCOPIC EXAMINATION 313 

notably spinach, when taken in large quantities, will give the stools a 
dark green appearance. Blackberries and huckleberries render stools 
dark brown or sometimes a greenish brown, due to the action of alkaline 
contents of the intestine upon the red coloring matter. Drugs may 
influence the color, bismuth turning the stools black, while after the 
use of iron, a grayish-black appears upon standing. After taking large 
doses of calomel, a green color may be seen, due to the presence of 
biliverdin which has been hurried through the intestinal tract before 
conversion to hydrobilirubin has taken place. With a diet which is 
liberal in fats, the stool may be pale yellow or even white while a clay- 
colored stool is seen in obstruction of the common duct when no bile 
gains access to the intestinal canal and when the fat has been insuffi- 
ciently digested. A light color is also seen in tuberculous and myeloid 
disease of the intestine and in pancreatic disease. Black, tarry stools 
frequently contain blood from the stomach or upper intestinal tract. 
In such cases, there is ample opportunity for the conversion of the 
blood to acid hematin or other hemoglobin pigments, but when the 
hemorrhage is lower in the intestinal tract, it is usually impossible for 
this conversion to take place. Streaks of blood seen on the surface of 
a formed stool usually have their origin in the rectum or anus. 

Odor.— The characteristic normal odor is said to be due to indol and 
skatol. The disagreeable odor is probably due in part to hydrogen 
sulphide. In fermentative dyspepsia, the stools may have a sour odor. 
The odor is offensive when the food is imperfectly digested. 

Mucus.— Normally, very small amounts of mucus are seen. Larger 
amounts are found in various pathological conditions, and are indica- 
tive of intestinal catarrh. When thoroughly mixed with the feces, 
the source is probably the small intestine, while larger quantities, 
which are not so well mixed, come from the large intestine. Occa- 
sionally stools which are almost purely mucus are seen in dysentery, 
intussusception, and ileocolitis. In such cases there is usually more or 
less blood. At times, especially in mucous colitis, the mucus may 
form casts of the intestines, or may appear in ribbons or shreds. 

Blood and Pus.— The significance of changed and unchanged blood 
has been referred to under the paragraph which discusses the color. 
Pus may be seen in ulcerative conditions of the bowel, due to syphilis, 
tuberculosis, dysentery, etc., or in larger quantities after a rupture of 
an abscess (e. g., appendiceal abscess) into the intestinal canal. 

Concretions.— The search for concretions is of especial importance 
after an attack of suspected biliary colic. The search for stones should 
be kept up for a period of several days after a suspected attack of biliary 
colic. The stool should be strained through a sieve. Several types of 
more or less complicated sieves are on the market, but they offer no 
especial advantage over a simple flour sifter or even the still simpler 
sieve used in the household for washing vegetables. The stool 
should be placed in the sieve and should be forced through with the 
aid of running water. The stones may be of the facetted, spherical or 



314 THE EXAMINATION OF THE FECES 

the mulberry types. "False gall-stones" are encountered at times 
after the administration of olive oil. They are merely masses of soap. 
Their formation has been used as a means of demonstrating the 
efficiency (?) of various fraudulent patent medicines (containing olive 
oil) sold to cause the passage of the stones. Confusion may be caused 
by seeds, olive pits, cherry stones, and small particles of impacted feces. 
For identification the stone should be ground up and chemical tests 
applied for the recognition of bile pigments and salts and cholesterol, 
the substances which compose the majority of gall-stones. (See page 
233.) 

Other concretions may be found. Coproliths are large, hard masses 
produced by the inspissation of feces in the intestines or appendix. 
True enteroliths are smaller and consist of an organic nucleus com- 
posed of blood clots, vegetable material, or similar substances, around 
which have been deposited layers of phosphates and intestinal detritus. 

Parasites.— An entire parasite may be found in the stools. The 
hookworm and the pinworm are notable examples. Segments of 
the tapeworm may be found. The search for the parasites may be 
aided by the use of the stool sieve as described for the search of con- 
cretions. "When an examination of the feces for parasites is con- 
templated, a vermifuge should be given followed by a vigorous purge. 
Embarrassing mistakes are sometimes made in the attempted diagnosis 
of parasites because of the confusion created by vegetable remnants 
which may resemble some of the smaller parasites, bits of orange and 
grape-fruit pulp having been taken for hookworm. Indeed, fly larvae 
have been mistaken for hookworm. When search is made for oxyuris, 
fecal material may be obtained from around the anus. Sometimes 
the worm can be seen just outside the anal orifice. The expulsion 
of taenia necessitates fairly drastic treatment. Full doses of a vermi- 
fuge should not be given merely for diagnostic purposes. Hensel, 
Weil and Jelliffe recommend administering only one-third of the 
therapeutic doses of the extract of male fern. 

Curd-like Masses.— Curd-like masses are sometimes of importance 
on account of their resemblance to other bodies (pus, concretions, etc.) . 
Casein will become tough and fibrous upon immersion in 10 per cent, 
formalin. Lumps of fat may present a curd-like appearance and can 
be identified by their solubility in ether and by the fact that they stain 
red with Sudan III. 



CHEMICAL EXAMINATION. 

Reaction.— The stool is mixed in a mortar with water to form a watery 
paste and the reaction is taken with litmus paper. Normally it is 
neutral, faintly acid, or faintly alkaline. 

Occult Blood.— Precautions should be taken to exclude the presence 
of iron, chlorophyl, meat derivatives, and mercury, any of which may 



CHEMICAL EXAMINATION 315 

give a positive reaction with benzidine. It is advisable to place the 
patient on a special diet from which are excluded all articles containing 
these substances. At the beginning of the diet a capsule containing 
0.3 gm. of powdered carmine should be given. The appearance of red 
coloration marks the first stool of the diet. The second or third 
specimen passed after this, is taken for the occult blood test. 

Benzidine Test.— A little of the stool should be rubbed up in a mortar 
or test-tube with enough water to form a paste of moderate con- 
sistency. A particle about the size of a hazel-nut meat is sufficient. 
With this is mixed thoroughly 1 cc. of glacial acetic acid and 2 or 3 cc. 
of saturated alcoholic solution of benzidin. After thorough mixing, 5 cc. 
of fresh hydrogen peroxide are added and the tube is corked and inverted 
several times. A Prussian-blue color indicates the presence of blood. 

Compressed tablets of benzidin may be used with a water emulsion 
of feces according to the method of Roberts (see Urine, page 237) . 
This method commends itself when the test must be performed in the 
patient's home or where the resources of the laboratory are not avail- 
able. 

For clinical purposes the benzidin test is preferred over the guaiac 
test, not only on the practical grounds that the benzidin solution may 
be kept on hand while the solution of guaiac resin must be prepared 
each time, but particularly because it is more delicate (5 to 10 times) - 1 

Guaiac Test.— A thick paste is made of the feces by grinding up a 
portion in a mortar or test-tube with a little water. One cc. of glacial 
acetic acid is stirred into this paste. As much ether is added as there is 
acidified fecal material. The contents are shaken and the ethereal 
layer is allowed to separate. Separation can be hastened by centri- 
fugalizing the mixture. The supernatant ethereal extract is poured 
into another tube. 2 

Significance.— The significance of blood in the stools has been dis- 
cussed in the preceding paragraphs dealing with the color of the stools. 

Bile Pigments.— Corrosive Sublimate Test.— A little of the feces is 
ground up in a mortar. A mixture is made with equal quantities of 
fecal paste and a saturated solution of mercuric chloride, and allowed 
to stand over night. Normally, only a red color develops, which is 
due to the presence of hydrobilirubin. A green color indicates the 
presence of unchanged bilirubin, which is pathological. Where the 
flow of bile has been obstructed completely, no color change appears. 

1 For a discussion comparing the various test, see Kelly, T. H.: Detection of Small 
Amounts of Blood, Jour. Lab. and Clin. Med., 1916, i, 897. 

2 This may be used for spectroscopic examination, detecting blood by the presence of 
the absorption bands of acid hematin. When properly diluted, a solution of acid hema- 
tin shows four absorption bands, one each in the red and yellow, and one between yellow 
and green and one between the green and blue. To it are added 10 drops of a freshly- 
prepared alcoholic solution of guaiac resin (1 gm. resin dissolved in 60 cc. of 95 per cent, 
alcohol) , and then either 20 or 30 drops of ozonized turpentine or 5 cc. of fresh hydro- 
gen peroxide. Ozonized turpentine can be prepared by allowing turpentine to stand 
for some time in an unstoppered bottle. A blue color appears in the presence of blood. 
Pus constitutes a possible source of error. 



3lf> THE EXAMINATION OF THE FECES 

Zinc Chloride Test. A small portion of feces is rubbed up in a mortar 
with a little alcohol acidified with a few drops of sulphuric acid. The 
acidulated extract is evaporated at about 50° C. to small bulk and is 
then shaken up with chloroform. The chloroform extract is separated 
from the residue by standing, or better by centrifugalization, and to this 
extract is added a few drops of concentrated solution of zinc chloride. 
A green fluorescence appears in the presence of hydrobilirubin. 

Significance of Findings.— The color of the stools is due to the 
presence of hydrobilirubin, which is formed by the reduction of the 
bilirubin furnished by the bile. Hydrobilirubin therefore should be 
found in normal stools and its absence points to occlusion of the 
common bile duct. Bilirubin, on the other hand, does not appear in the 
normal adult stool and its appearance points to insufficient reduction 
due to too rapid peristalsis, as in diarrhea, or dysentery. 

MICROSCOPIC EXAMINATION. 

Suspicious portions of the stool are picked out with wooden appli- 
cators or tooth-picks and placed on slides for examination, or a 
portion of the stool thoroughly mixed with water to form an emul- 
sion. A portion about the size of a hazel-nut is transferred to a 
mortar, and is triturated with enough w T ater to form a thin paste. 
Drops are then transferred to slides and subjected to treatment with 
various reagents. For examining feces, large object slides (2 by 3 
inches) are convenient, giving a large area for study. The directions 
which have been given in describing the microscopic examination of 
both urinary sediments and gastric contents should be observed here. 
In brief, the field should be well darkened. 

Slides may be treated as follows : 

1. One drop is covered with an object slide and is examined in turn 
with the low r and the high-power dry lenses. The number and condi- 
tion of the muscle fibers should be noted, paying especial attention to 
the presence or absence of striation. One should also look for parasites, 
ova, red blood cells, mucus particles, fatty acid crystals, salts of cal- 
cium, drops of neutral fat, and food remnants. 

2. The next drop is treated with one or two drops of saturated solu- 
tion of Sudan III (made with 70 per cent, alcohol) and is covered with 
a cover-slip. The fat droplets stain red. 

3. To this droj) is added a drop of Lugol's solution. After thorough 
mixing, it is examined for starch cells, cysts of endameba, yeast-cells, 
and fungi. 

4. To the last drop is added 36 per cent, acetic acid. The prepara- 
tion is heated gently. The food masses are broken up, the soaps are 
converted to fatty acids, and connective tissue is rendered transparent. 

Undigested Food Remnants.— Th e food remnants which may be seen 
in the feces include meat fibers, connective-tissue fibers, fat droplets, 
fatty acid crystals, or soaps, plant cells, starch granules, and detritus. 



MICROSCOPIC EXAMINATION 



317 



Practically all formed elements in the feces are colored yellow or 
brownish-yellow unless, of course, the flow of bile has been shut off. 

(Fig. 81.) 




Fig. 81. — -Collective view of the feces. (Eye-piece, III; objective 8 A, Reichert.) 
a, muscle fibers; b, connective tissue; c, epithelium; d, white blood corpuscles; e, spiral 
cells; /, fitted ducts; g, cork cells; h, parenchyma with starch; i, plant hair; k, triple- 
phosphate crystals in mass of various microorganisms; I, stone cell. (v. Jaksch.) 

Meat fibers appear as more or less cylindrical bodies with rather 
square ends. When digestion is not complete, cross-striation may be 
made out. Connective tissue is recognized by the reticulum of white 
fibers. Vegetable and 'plant cells may occur in a variety of forms. Only 
experience will enable the worker to familiarize himself with the 
different types. Reference to the illustration will serve to present some 
of the more common types including stone cells, spiral vascular tubes, 



&vm 



mmm 




Fig. 82. — Crystals of fatty acids, a, mingled with mucus; b, around fat droplets in an 
infant's stool to which glycerin has been added. (From Schmidt and Strasburger.) 



vegetable hairs, large potato cells, and the vegetable cells containing 
chlorophyl which the inexperienced are so apt to mistake for parasitic 
ova. Fat is seen in the form of refractile globules of varying size which 
stain red with Sudan III. Fatty acid crystals are seen (Fig. 82). 
They are delicate pointed needles, which melt with heat to form globules 
and may be identified by their solubility in cold alcohol. The soaps 
appear in the form of somewhat coarser needles, The greater pro- 



318 THE EXAMINATION OF THE FECES 

portion of the soaps are calcium salts, which are insoluble in alcohol 
though the salts of the alkaline metals are soluble in this reagent. The 
soaps may also appear in the form of irregular scales, colored yellow 
with biliary pigments (Fig. 83) . Small particles of casein or coagulated 
albumin may be seen, also colored yellow. 

The microscopic examination is of great importance from the 
standpoint of detecting parasitic ova, and the student should have 
ample practice in examining normal feces if for no other reason than to 
be able to recognize vegetable cells when he sees them, so that he will 
not confuse them with parasitic ova. 



Fig. 83. — a, fatty acid amorphous masses; b, soap crystals. (From Schmidt and 
Strasburger.) 

Red Blood Cells.— Red blood cells may be seen in the feces, when the 
loss of blood is great or when the hemorrhage is from a point low in the 
intestines. Blood shed in the stomach, small intestines, or upper 
portion of the large bowel is usually hemolyzed. Red blood cells may 
be microscopically identified by the characteristics which have been 
described in the chapter on Urine (see page 229) . 

Leukocytes.— Conditions which lead to the appearance of leukocytes 
in the feces have been referred to previously under the discussion of the 
gross appearance. 

Mucus.— Mucus appears in more or less homogenous or stringy 
masses with faint contours. Upon addition of acetic acid, precipita- 
tion occurs, so that irregular linear markings appear. The condition 
in which mucus is seen in the stools have also been discussed in the 
paragraph on the gross appearance of stool. 

Crystals.— Crystals in the feces have little significance. Triple 
phosphates crystals are occasionally seen especially when the specimen 
has been allowed to decompose partially or has been mixed with urine. 
Charcot-Leyden crystals are found with many of the intestinal para- 
sites. Stitt states that while they are practically always absent 
from bacillary dysentery stools, they are not infrequently found in 
stools containing ameba. Crystals of fatty acids and soaps have been 
discussed. Cholesterol crystals are rare (Fig. 84). Crystals of 
bismuth suboxide may be seen after the administration of bismuth 
salts (Fig. 85). They are dark, even black, in color and irregularly 



GENERAL BACTERIOLOGICAL EXAMINATION 



319 



rhombic in form. Occasionally hematoidin crystals are seen after 
hemorrhages. Calcium oxalate crystals are found at times. 







Fig, 84. — Cholesterin crystals. (Simon.) 






■;?km 









sv-'o'/:- 



Fig. 85. — -Sulphide of bismuth crystals in the feces, (v. Jaksch.) 

Search for Parasitic Ova.— This may be conducted with slides pre- 
pared directly from the stool or from the watery emulsion of the stool. 
At times, however, ova cannot be found when there is every reason to 
believe that they are actually present. In such cases, it is desirable 
to use some method for concentrating the ova and for freeing the 
stools of other substances. Methods are given in the section of this 
chapter which deals with intestinal parasites. 



GENERAL BACTERIOLOGICAL EXAMINATION. 

For direct study, the bacteria are separated by centrifugalization. 
A small portion of feces is ground up in a mortar, as for the micro- 
scopic examination. The emulsion is placed in a centrifuge tube and 
centrifugalized for a short period of time. The bacteria remain in the 



320 THE EXAM IX ATI OX OF THE FECES 

supernatant clear fluid, while the solid portion goes to the tip of the 
centrifuge tube. The supernatant fluid is poured into another centri- 
fuge tube and to it is added twice its volume of 95 per cent, alcohol. 
This is centrifugalized and a sediment is obtained which contains the 
bacteria. The sediment is taken up with a pipette and placed upon 
a slide. After an even spread is made, it may be dried, fixed and 
stained with Gram's stain. The organisms will appear as Gram- 
positive (/. c, are purple in color), or Gram-negative. In normal 
adult stools the cocci will predominate over the Gram-positive bacilli. 
The Gram-positive bacilli are more numerous on a meat diet. 

Detection of Tubercle Bacilli in the Feces.— Blood stained bits of feces 
or bits of mucus should be selected and smears made. The smears 
should be stained with carbol-fuchsin and decolorized with especial 
care to rule out smegma bacilli, as directed in the chapter on Urine. 

Another method is to treat the feces as directed in the following 
paragraphs on the "Method of Isolating Tubercle Bacilli from the 
Feces" up to the point where the sediment is ready to be inoculated 
into culture tubes. This sediment should be washed after centri- 
fugalization by pouring off and discarding the supernatant fluid. To 
the sediment add absolutely fresh and sterile distilled water. After 
mixing the sediment thoroughly to secure the solution of the salts 
which it may contain, the tube is centrifugalized again, the supernatant 
fluid is decanted, and the sediment is placed on a slide. It may be 
necessary to add a drop of Mayer's egg-albumen to make the sediment 
adhere to the slide. 

The search for tubercle bacilli in the stools is far from satisfactory. 
Even when acid-fast bacilli are found, there is room for doubt as to 
whether the bacilli are tubercle bacilli or smegma bacilli, and as to 
whether they originated from the intestinal tract or were swallowed 
with sputum. 

Method of Isolating Tubercle Bacilli from the Feces.— The feces are 
collected in wide-mouthed jars or bottles, and are diluted with three 
volumes of freshly distilled water. The material is mixed thor- 
oughly with the water and filtered through several thicknesses of 
gauze to remove the solid particles. The filtrate is saturated with 
sodium chloride and allowed to stand thirty minutes, at the end of 
which time there will be found a floating film containing all the bacteria. 
This film is scooped up with a spoon and placed in a wide-mouthed 
bottle with an equal quantity of normal sodium hydrate. The bottle 
is stoppered, shaken vigorously, and then kept at a temperature 
of 38 c C. for three hours. During this time it is shaken frequently, 
and at the end of the time it is neutralized to litmus with normal 
hydrochloric acid. This is centrifugalized and the sediment inoculated 
into several tubes of Petroff's or other suitable medium. 

Other Bacteria in the Feces. -More extended bacteriological study 
of the feces requires special media and the resources of a trained bac- 
teriologist; the methods do not lend themselves well to execution in a 



SCHMIDT AND STRASBURGER'S TEST DIET 321 

small clinical laboratory. Therefore, only directions for the detection 
of organisms of the typhoid and colon groups will be given here. 

Isolation of Typhoid Bacilli.— The feces are obtained in a clean vessel 
and as free from outside contamination as possible. Two or three 
loopsful are taken up with a bacteriological loop and mixed with 
nutrient bouillon to make a dilute emulsion. If the stool be fluid, 
dilution is unnecessary. Thoroughly hardened Endo's or Krumwiede's 
brilliant green medium, or eosine-methylene-blue agar, is streaked and 
the plates incubated from twenty-four to forty-eight hours. A number 
of suspicious colonies (5 to 10) should be fished out with a platinum 
loop and inoculated into as many tubes of nutrient broth. After six 
hours in the incubator, hanging-drop slide preparations are made to 
determine motility. Transplants should be made from tubes which 
show motile organisms, into fermentation tubes containing sugar broths 
(including saccharose, mannite, dextrose and lactose), colored with 
neutral red. The typhoid bacillus forms acid with lactose, and causes 
no change with glucose, while the organisms of the colon group form 
acid and gas with both glucose and lactose. In the morning, if sugar 
readings are suggestive, agar slants should be inoculated. The growth 
on the slant may be used for agglutination test with a known typhoid 
serum in order to complete identification. 

SCHMIDT AND STRASBURGER'S TEST DIET. 

The patient is placed on Schmidt and Strasburger's test diet No. 2. 
This comprises a daily allowance of 1.5 liters of milk, 3| eggs, strained 
oatmeal gruel (prepared from 80 grams of oatmeal), 100 grams of 
zweibach, 20 gm. of butter, 20 gm. of sugar, 125 gm. of steak (weight 
before cooking), and 190 gm. of potato (weight before cooking). A 
capsule containing 0.3 gm. powdered carmine should be given when 
the diet is started. The second or third stool passed after the first 
appearance of carmine may be used for examination. 

For this test a Schmidt fermentation apparatus is used (Fig. 86). 
In the bottle are placed about 5 gm. of fresh feces, if of medium con- 
sistency. More or less should be taken if the specimen be very thin 
or very hard. When the stools are fluid, the bottle should be filled. 
The stool is moistened with water, with which it should be thoroughly 
mixed, the bottle is completely filled with water, and the rubber stopper 
inserted. The first of the twin tubes is then removed and filled 
with water. The rest of the apparatus is inverted over this tube, 
which is stoppered with the rubber stopper. The entire apparatus is 
then turned back so that the twin tubes occupy the inverted position. 
The apparatus is kept in the incubator at 37° C. for twenty-four hours. 
At the end of this time the height of column of water in the second twin 
tube is noted. The amount furnishes a guide to the amount of fer- 
mentation. The bottle containing fecal material is opened and the 
reaction of the contents is taken with litmus. 
21 



322 



THE EXAMINATION OF THE FECES 



Normal Findings.— Lnder ordinary conditions, little or no gas is 
found, and the reaction to litmus usually shows little change. It is 
distinctly abnormal if the last tube is filled more than one-half with 
water. An acid reaction to litmus and light color of the fecal mixture 
indicate carbohydrate fermentation. A dark color and alkaline reac- 
tion indicates putrefaction. 

Microscopic Examination.— The stool obtained after the test diet 
should be studied microscopically, as directed in preceding paragraphs. 
With normal digestion, there is found only a mod- 
erate number of muscle fibers, showing no distinct 
striation. With normal gastric digestion the 
number of connective-tissue fibers would be incon- 
siderable. Starch granules are not found in normal 
stools. Fat should be present only in small 
quantities and in the form of droplets or soaps is 
a sign of deficient fat digestion. With ordinary 
diet and normal digestion, all sorts of vegetable 
_C remnants may be seen. 



FINDINGS IN THE FECES IN DISEASES. 

Typhoid Fever.— There are no constant findings 
in typhoid fever. It is often stated that diarrhea 
is an early symptom. While this may be so in 
some localities, it is by no means invariable. 
Osier states that out of 1500 cases, 516 showed 
diarrhea and 249 constipation. As the disease 
progresses, diarrhea, when present, becomes 
aggravated and the stools take on the "pea-soup" 
appearance. During the course of the disease, 
careful watch should be maintained for the ap- 
pearance of blood on account of the ever-present 
possibility of hemorrhage. With a severe hemor- 
rhage, so-called " tarry stools" may be seen, which 
are composed almost entirely of blood. 
Dysentery.— The stools are small and frequent, containing much 
mucus, many red blood cells and leukocytes, and a little fecal material. 
In the bacillary form, it may be possible to isolate one of the strains 
of the dysentery bacillus, while in amebic dysentery the Endamceba 
dysenteriae may be found when the starch is conducted properly. 

Cholera.— Small bits of mucus are seen floating in a serous stool. 
The frequent and profuse stools are often referred to as "rice-water 
stools." Microscopically, mucus, epithelial cells, and numerous 
bacteria may be seen. The majority of the latter are comma bacilli. 
Acute Gastro-enteritis.— The stools are small and frequent, usually 
pasty in color, occasionally green. The fatty acids are often present 




Fig. 86.— Schmidt's 
fermentation tubes. 



FINDINGS IN THE FECES IN DISEASES 323 

in large amounts and undigested meat fibers may be found on micro- 
scopic examination. In severe cases, there may be blood and at times, 
an almost pure exudate of serum, leukocytes, and epithelium, 

Obstruction of the Common Bile Duct.— When the flow of bile into the 
intestine is prevented either by a catarrhal swelling of the lower portion 
of the common duct or by the lodgment of a calculus in the duct, the 
stools present the typical "clay-colored" appearance because of the 
absence of biliary pigments. The odor is often unusually offensive. 
When the patient is allowed to eat fats, the stool frequently has a 
distinctly greasy appearance and shows undigested fat. On micro- 
scopic examination, the absence of bile coloration in the food remnants 
and the presence of large numbers of fatty acid and soap crystals are 
found. Chemical tests show the absence of hydrobilirubin though if 
the obstruction be only partial, the stool may show a certain amount 
of coloration. In cases of catarrhal jaundice, it is worth while from a 
prognostic standpoint to examine the stools for the first return of 
hydrobilirubin, since this is sometimes detected before the jaundice 
begins to lessen. 

Pancreatic Disease.— The stools are often bulky and contain large 
quantities of undigested meat and fats. Barker states that there is 
more neutral fat than is seen in acholic stools. Microscopic examina- 
tion shows neutral fats, many fatty acid crystals, and meat fibers 
showing striation plainly. 

Acute Catarrhal Enteritis.— The stools vary in character depending 
upon the portion of the intestinal tract which is principally at fault and 
upon the severity of the process. The color depends upon the amount 
of bile, ranging from a paste-like gray color to a dark brown. The 
consistency is usually thin and watery though it may be like that of 
gruel. Small quantities of mucus are seen. If the small intestine be 
involved, the mucus will be bile-stained, while if the large intestine be 
the seat of the inflammatory process, the mucus will appear in clear 
masses, not stained with bile. Blood may be seen in small amounts. 
Portions of undigested food may be seen, especially when the small 
bowel is involved. Microscopically, epithelial cells, leukocytes, 
organisms in great number, and crystals may be found. 

Chronic Catarrhal Enteritis.— When involvement of the small intestine 
predominates, the stools often show bile-stained mucus and small 
particles of undigested food. When the colon is affected, the stools 
are thin and contain a considerable amount of clear mucus. At times 
constipation is seen instead of diarrhea. 

Mucous Colitis.— Large quantities of mucus are passed in the stools, 
sometimes comprising all of the material which is passed in a given 
movement. This may be seen in masses or as tubular casts. The 
patients often state that they have "passed a part of the bowel." 
Microscopically, the mucus nature is apparent. In the granular 
ground substance may be found cells, largely partially degenerated 



324 THE EXAMINATION OF THE FECES 

epithelial cells and leukocytes, with crystals of various sorts, occa- 
sionally including Charcot-Leyden crystals, and at times fine intestinal 
sand. In making a diagnosis it is important to remember that mucus 
in large quantities is also found at times in other pathological condi- 
tions, notably cancer of the bowel. 

Intestinal Ulcers.— As a rule, diarrhea is present, though this is not 
always the case. The three cardinal evidences of intestinal ulcer are 
blood, pus, and tissue shreds. 

Carcinoma of the Intestine.— There is nothing characteristic in the 
feces, though they may be frequent and sometimes show blood, pus, 
and mucus. 

Intussusception.— In obstruction of the bowel due to intussusception, 
the stools contain blood and mucus. There is also fecal vomiting. 

Thrombosis of the Superior Mesenteric Artery.— When acute, there is 
seen nausea, vomiting, and the passage of stools containing much blood. 

CHEMICAL EXAMINATION OF GALL-STONES. 

The suspected stone should be ground up and treated with a little 
hot water to remove the bile acids. The insoluble residue is treated 
with a warm mixture of equal parts of alcohol and ether. This serves 
to dissolve the cholesterol, and the alcohol-ether solution (extract A) 
is set aside to be tested for cholesterol. 

The insoluble residue remaining after the extraction with the alcohol- 
ether mixture may contain bilirubin and calcium salts (residue B). 

The ether-alcohol extract (extract A) should be evaporated carefully 
over a water-bath, or better, at room temperature. If cholesterol be 
present characteristic rhomboidal crystals will form. These may be 
placed upon a slide and identified microscopically. After covering the 
crystals with a cover-glass, concentrated sulphuric acid may be run 
under the cover. The crystals soften a little at the edges and become 
carmine-red in color. When Gram's iodine solution is added, the color 
changes to blue, red, green, or violet. 

The residue B may be tested for bilirubin. First a small quantity 
of hydrochloric acid should be added. This sets the bilirubin free by 
breaking down the calcium compounds. Chloroform is added to 
extract the bilirubin. The chloroform extract is then tested for bile- 
pigments by the tests for bile already described in the chapter on 
I rine. 

INTESTINAL PARASITES. 

Method of Search.— The search for parasites is conducted according 
to tin-size and habits of the species in question. With the tapeworms, 
segments of the adult worm should be looked for in the stool. Oxyuris 
vermienlaris may be found in scrapings taken from about the anus. 
With certain other species, the adult parasite is not found in the stool or 



INTESTINAL PARASITES 325 

is found only with difficulty, and reliance must be placed upon a search 
for ova. In still other cases, ova are not found at all, adults are found 
only occasionally, and reliance must be placed on finding the larvre 
in other tissues (Trichinella spiralis) . 

The search for ova may be conducted with slides prepared directly 
from the fresh stools or from a watery emulsion of the stools, using a 
well-darkened microscopic field. This method is satisfactory in the 
majority of cases. At times, however, it is unsuccessful and in such 
cases the stools should be concentrated. Two methods are given here. 

Method of Dock and Bass.— A portion of the feces is diluted to 10 times 
its volume with water. The suspension is filtered through 2 or 3 
thicknesses of gauze in a funnel to remove the coarser food-remnants, 
especially vegetable remains. The filtrate is then centrifugalized for 
a time, long enough to bring the ova to the tip of the tube but not long 
enough to bring down all the food particles or bacteria. The exact 
time in a given centrifuge can only be ascertained by trial. Dock and 
Bass found that the time with the Purdy centrifuge was about four 
seconds. The supernatant fluid is poured off and discarded. The 
original volume is restored by adding water. Centrifugalization is 
repeated in the same way and the supernatant fluid is again poured off. 
The process is repeated a third time, after which the sediment is ready 
for examination. 

Method of Yaoita. — By this method everything in the stool except 
certain resistant food elements and ova is dissolved and the sediment 
examined in the ordinary way. 

Take from different portions of the stool about 5 small particles, 
each the size of a pea, and place in a test-tube with about 10 to 15 cc. 
of a mixture of equal parts of 25 per cent, antiformin and ether. 
Shake vigorously. There will be a marked evolution of gas. If the 
feces are hard, an emulsion should be made by stirring them with 
a glass rod, adding antiformin, and applying heat if necessary. Ether 
is then added. The fluid is now filtered through a layer of loose gauze 
to remove the larger food remnants and the filtrate centrifugalized for 
one minute. Four distinct layers are seen. The upper one is ether, 
colored yellow by the dissolved neutral fats and fatty acids. The 
second is a ring with suspended fine food particles, while the third is 
antiformin, colored yellow-brown or blackish-brown, containing detritus 
and dissolved food material. The lower layer, occupying only a por- 
tion of the tip of the centrifuge tube, contains undissolved food material 
(cellulose, epithelium, salts, elastic tissue, muscle fibers) and parasitic 
ova. The latter are affected little or not at all by the reagents. Fifty 
per cent, antiformin has a deleterious effect upon the eggs. 

The dimensions of the parasites and ova given throughout the 
chapter have been taken from the work of Fantham, Stephens, and 
Theobald. 

Examination for Ameba.— In making a search for amebse, the stools 
should be obtained in a fresh condition. The examination should be 



320 



THE EXAMINATION OF THE FECES 



conducted at the bedside when possible. It is advisable to have the 
stool passed into a warmed vessel containing a little warm normal salt 
solution. A bit of fecal material, preferably a particle of mucus, should 
be placed on the slide for study, with a hair between the slide and 
cover-slip, to prevent crushing the organisms or interfering with their 
locomotion. If the room temperature be below 70 or 75° F., a warm 
stage should be employed. An electrically heated warm stage, 
equipped with an automatic thermoregulating device and provided 
with a thermometer, is convenient. This should be kept at body 
temperature. 

Entamoeba Histolytica (Syns. : Ameba coli; Ameba dysenteric).— 
The parasite is found in the stools of patients suffering with amebic 
dysentery and in the pus from liver abscess associated with dysentery. 
Its average size is from 25 to 35ju. The eccentric and often feebly 




refractile nucleus is from 4 to Q/x in diameter. It is surrounded 
by coarsely granular body-substance which is divided into endoplasm 
and highly refractile ectoplasm. In the body of the parasite are found 
numerous foreign bodies, epithelial cells, bacteria, leukocytes, red blood 
cells, etc. Pseudopodia, composed chiefly of the ectoplasm, are pro- 
jected by the organism. Smaller encysted forms are seen occasionally 
(so-called tetragena forms). Their ectoplasm is seen only when 
pseudopodia are projected. Red blood cells may be seen in the endo- 
plasm. Nuclear division occurs, some of the round cysts containing 
four nuclei (tetra-nucleate cysts). Demonstration of the cysts is 
aided by emulsifying the stool with tincture of iodine, which colors the 
cyst red. Diagnosis is not possible without demonstration of ameboid 
motion. This can be observed only in fresh warm specimens. Stitt 
recommends staining the living ameba by tinging the fecal emulsion 
with a 1 per cent, aqueous solution of neutral red. 



INTESTINAL PARASITES 327 

The geographic distribution is wide. It is a frequent cause of dysen- 
tery in the tropics and in the southern portion of the United States. 
Even in the northern states and in Europe occasional cases are seen. 

Entamoeba Coli (S.yns. : Ameba coli; Entameba hominis). — This 
harmless parasite is found not infrequently in the stools of normal 
individuals. It lives as a commensal in the upper part of the digestive 
tract, where the feces are still of thin consistency. With increase in 
the consistency of the fecal material arid the change in reaction which 
takes place in the lower bowel, the organism becomes encysted, but 
when the stools of infected individuals are loose, ameboid forms may 
be found. 

The ameboid form measures from 26 to 30/x in diameter. Ameboid 
movement is not marked. No distinction can be made between 
the endoplasm and the ectoplasm. The central' y placed and sharply 
denned nucleus is rich in chromatin. Ingested red blood cells are 
rarely seen. The encysted forms are larger, showing eight nuclei instead 
of the four seen in the encysted stage of the pathogenic E. histolytica. 

For the purpose of differentiation between the two forms, the follow- 
ing table has been given by Walker: 

MOTILE STAGE. 

Entamoeba histolytica. Entamoeba coli. 

Appearance hyaline. Appearance porcellaneous. 

Refractiveness more feeble. Refractiveness more pronounced. 

Movements active in the fresh stool. Movements sluggish. 
Nucleus more or less distinct. Nucleus distinct. 

Chromatin of nucleus scanty. Chromatin of nucleus abundant. 

ENCYSTED STAGE. 

Entamoeba histolytica. Entamoeba coli. 

Cyst smaller. Cyst larger. 

Cyst less refractive. Cyst more refractive. 

Cyst usually contains elongated Cysts do not contain "chromidial 

refractive bodies, "chromidial bodies." 

bodies." 

Nuclei never more than 4. Nuclei 8 or more. 

Cyst wall thinner. Cyst wall thicker. 

Trichomonas Intestinalis (Syn. : T. hominis) . — This flagellate has a 
pear-shaped body, measuring 3 to 4/i in breadth and 10 to 15/* in length. 
The posterior end terminates in a point. An undulating membrane 
terminates in a posterior nagellum, and three longer flagella projecting 
from the anterior end. A cytostome may be seen near the nucleus 
(Fig. 88). The organism lives in any portion of the intestinal tract 
in which the contents have an alkaline reaction. The pathogenic role 
of this parasite is disputed. 



328 



THE EXAMINATION OF THE FECES 



'/'. vaginalis is the term applied to an apparently identical form which 
is seen in the vagina and occasionally in the urine. The present 
opinion is that T. intestinalis and T. vaginalis are the same, though it is 
admitted that the form seen in the urine and vagina is larger and not so 
definitely pear-shaped. 




Trichomonas intestinalis. (Brumpt.) 



Lamblia Intestinalis (Syns. : Cercomonas intestinalis, Megastoma 
entericum, Lamblia duodenalis). — This bilaterally symmetrical and 
pear-shaped organism is 10 to 21/x in length and from 5 to 12/x in 
width. A striking point is the anterior oblique depression which 
serves as a sucking disc by which the parasite attaches itself to the 
intestinal epithelial cells. The organism is covered by a thin cuticle 
(Fig. 89). There are four pairs of flagella, the median pairs being the 




(Brumpt.) 



more active. The nucleus, which is situated in the thinner, anterior 
portion, is at first dumb-bell in shape but later separates to form two 
nuclei, one on each side. Two long longitudinal ridges extend from the 
sucking disc to the posterior extremity. In the flagellated form the 
parasite is found in the jejunum and duodenum. As it passes into the 



IN TES TIXA L PA RA SI TES 



320 



larger intestine, it becomes encysted. Oval cystic forms with thick 
cyst-wall are seen in the. stools. L. intestinalis is found associated with 
many cases of severe diarrhea, and is thought to be the causative agent. 
Balantidium Coli (Syn.: Paramecium coli). — The body of this 
ciliate is oval, measuring from 50 to 70m in breadth and from 60 to 100/x 
in length. The cilia are especially marked about the peristome, which is 
situated at the anterior end. Ectoplasm and endoplasm are separated, 
the former forming a longitudinally striated cuticle and the latter 
containing granules of starch, bacteria, drops of fat and mucus, and 
occasionally red and white blood corpuscles. At the posterior extrem- 
ity there is an anal opening (cytopyge). A macronucleus and a 
micronucleus are seen (Fig. 90). Propagation is by transverse division. 
Encystment takes place under unfavorable conditions. Reports of 
balantidium infection indicate a fairly wide geographical distribution. 
Cases have been observed in America. Walker has reported an 









Fig. 90. — Balantidium coli: 1, 2, division; 3, conjugation. (After Leuckart, from 
Doflein.) 



extensive study from the Philippines. These ciliates are parasitic for 
hogs and other animals as well as for man. In man, they are evidently 
the cause of a severe and occasionally fatal diarrhea. 

Balantidium Minufcum. — This parasite has also been reported para- 
sitic for man. It measures about 20/* long by 14 to 20/x broad. The 
peristome extends to the center of the body and resembles a fissure. 

Trematodes or Flukes.— The trematode worms or flukes are non- 
segmented, flat, leaf-shaped parasites. 

Fasciola Hepatica.— There are two suckers, one at the anterior end, 
the oral aperature, and one adjacent to it on the ventral surface, the 
ventral sucker. The dimensions are, length about 20 to 30 mm., 
breadth 8 to 13 mm. Spines are seen on both dorsal and ventral 
surfaces. It is a hermaphrodite. The ovary is near the anterior end 
and the testes, which ramify greatly, occupy a large portion of the 



330 THE EXAMINATION OF THE FECES 

body. The uterine rosette is in the anterior portion of the body. 
The intestine bifurcates and ramifies. The eggs are oval, yellowish- 
brown in color, are provided with lids (opercula) and resemble the ova 
of Dibothriocephalus latus, except that they are much larger, averaging 
about 70 to 132/t. 

The liver fluke inhabits the biliary passages of herbivorous animals 
and is of economic importance on account of the fact that it infects 
sheep and produces what is termed "liver-rot," thereby causing great 
loss. Only occasional cases have been reported in man and in most of 
these the infection has been rather mild, having been discovered in 
some instances only at autopsy. Diagnosis can be made during life 
only by finding the ova in the stools. The parasite is found in Europe, 
and has been reported in North and South America, North Africa, 
Australia and Asia. The intermediary host in which development of 
the larva (miracidium) takes place, is the snail. 

Opisthorchis Felineus (Syns.: Distoma conus, Distoma lanceo- 
latum, Distoma sibiricum). — The cat-fluke usually measures from 1.5 
to 2 mm. by 8 to 1 1 mm. and is yellowish-red in the fresh condition. The 
flat body shows a conical neck. There are two suckers. The posterior 
end is either rounded or pointed. The oval eggs, measuring 11 by 
30ju, show a clearly defined lid at the pointed pole. This species 
inhibits the biliary ducts and gall-bladder of the domestic cat, and is 
also found in the dog, fox, and in other animals. It is found in man, 
where extensive changes may take place in the liver. Numerous cases 
have been reported from Siberia. The intermediate host is in certain 
fish and the infection is presumably acquired by eating uncooked fish. 

Clonorchis Endemicus (Syn. : Distoma japonicum).- — Within this 
genus are probably two species, C. endemicus and C. sinensis. The 
differentiation has not been clearly made, however, and the existence 
of two species is denied by some. The genus Clonorchis comprises the 
more important human liver-flukes. In shape the flukes resemble the 
opisthorchis felineus, but have been separated because the testicles 
branch while those of Opisthorchis are lobed. The fluke is common 
in Japan and China and is diagnosed by the presence of eggs in the 
stools. The ova measure about 13 to 16ju by 26/*, and show sharply 
defined lids. The exact mode of infection is not definitely known, but 
it is felt that a mollusc is the primary intermediate host and a fish 
the secondary intermediate host. 

Fasciolopsis Buski (Syn. : Distoma buski). — This thick, brown fluke 
has a breadth of from 5.5 to 14 mm. and a length ranging from 24 
to 70 mm. The ovary and shell-gland are in the center of the body 
with the testes behind and the uterus in front. The ventral sucker is 
three or four times the size of the oral sucker. The eggs measure from 
77 to 88ju in breadth and from 120 to 140^ in length. They are thin- 
shelled and show a small lid. F. buski lives in the intestine of pigs, 
occasionally in man. In man, it is found in Siam, India, Assam, 
Sumatra, and China, and is particularly common in Cochin China. 
The larval stage is said to take place in shrimps. 



INTESTINAL PARASITES 331 

Genus Schistosomum.— The genus Schistosomum differs from the 
other flukes in that the sexes are separate. The bodies of the male 
widen behind the ventral sucker, and the lateral portions roll ven- 
tral-wards to form a partially closed canal (canalis gynecophorus) , 
within which the female lies. The female is slender and filiform, about 
20 mm. long, while the male is 12 to 14 mm. long. The eggs are oval 
and somewhat elongated, measuring about 40 to 60^ in breadth by 120 
to 150/x in length. They do not have lids, but show either lateral or 
terminal spines. They are called blood-flukes, since they invade the 
circulatory system. Infection with the blood-flukes is termed bil- 
harziasis since Bilharz was the first to discover the parasite and describe 
the disease. The early development of the worm takes place in the 
liver. The young worms travel from this point in the portal veins 
and its tributaries against the blood stream. Symptoms frequently 
noted are due to bladder involvement, such as hematuria and vesical 
irritation and those due to lodgment of the worms in the hemorrhoidal 
veins, such as "bleeding piles" and diarrhea. The eggs are found in 
the urine and in the feces. There are three human species. 

Schistosomum Hematobium (Distoma Haematobium Bilharz).— 
The male shows four or five large testes. The gut stem is short. In 
the female, the ovary is in the posterior half of the body and the uterus 
is long and voluminous. The ova show terminal spines, have no lids, 
and measure 40 to 60^ in breadth and 120 to 150/x in length. The 
geographical distribution covers many regions in Africa while isolated 
cases have been reported from Cyprus, Greece, India, and Arabia. 

Schistosomum Mansoni.- — In this form, the eggs show lateral spines. 
The male has eight small testes and the gut-stem is very long, while 
the ovary of the female is in the anterior half of the body and the 
uterus is short. This species is said to be found only in the Congo, 
in the West Indies, in Brazil, and in the Southern States of this country. 

Schistosomum Japonicum. — This species differs chiefly from the 
preceding ones in the fact that the skin of the adult worm is not 
tuberculated. The lower part of the male infolds to a greater extent. 
There are six to eight irregularly elliptical testes. The eggs have small 
lateral spines or thickenings, and opposite them cap-like thickenings 
have been described, though Stitt states that there are no spines. 
The embryos hatch out in the water in a very short time, and will live 
there for twenty-four hours. The "larvae" which are formed penetrate 
the skin and in some unknown way reach the portal veins. The pene- 
tration of the skin is marked by itching of the legs. The embryos also 
develop in molluscs. This species occurs in China, Japan, and the 
Philippines. Cattle, dogs, and cats, are infected as well as man. In 
man, the effects produced are anemia due to loss of blood, ascites, 
phlebitis and thrombosis, bloody diarrhea, etc. The eggs are readily 
found in the feces. 

Class Cestoda.— The flat worms are divided in the two classes, 
Trematoda and Cestoda. The Trematoda, or flukes, have been dis- 



332 THE EXAMINATION OF THE FECES 

cussed in the preceding paragraphs. The Cestoda comprise the tape- 
worms. They have been compared by Stitt on anatomical grounds to 

a series of individual flukes united in a ribbon-like colony. The adult 
worm is composed of a scolex or head and of a number of proglottides, 
()]• segments. The scolex serves to attach the worm to the host. 
The head is provided with a sucking apparatus by means of which it 
fastens itself to the intestinal mucosa. In addition, some species are 
provided with hooklets. When present, they are carried on the 
rostellum. The scolex is divided into head and neck. The segments 
are formed from the scolex, so that the scolex actually produces the 
colony of which it is the head. The youngest segment is that nearest 
the scolex and the oldest that at the distal end of the colony. The 
number of segments varies, T. echinococcus having only three or four 
and T. saginata having about 2000. The cestoda are hermaphrodites, 
and each segment contains ovaries, cirrus, uterus, vasa deferentia, 
genital pores, yolk glands, etc. The proglottides are covered with 
elastic tissue and absorb their nourishment through the surface. 

For the development of the embryo from the eggs formed in the 
segments, passage through an intermediate host is necessary. The 
host is different for each species. The eggs leave the host which 
harbors the adult-worm either free in the feces or contained in a 
detached segment. Their entry into the intermediate host is purely 
passive. After ingestion, the embryonic envelope is dissolved in 
the alimentary tract and the six-hooked embryo or oncosphere is set 
free. This bores through the gut and encysts itself in the body tissues. 
Here the hooklets are discarded and a bladder-like structure is formed in 
which the scolex is inverted. When tissue containing this cyst is eaten 
by a suitable host, the cyst wall is dissolved and the liberated scolex 
attaches itself to the intestinal wall and develops a series of pro- 
glottides. The embryo of Dibothriocephalus latus, however, does not 
form a cyst. In most instances, the adult stage is that seen in man, 
T. echinococcus being an exception. The larval stage takes place in 
different animals. 

From a practical standpoint, two points are worthy of notice. One 
is that while segments may be detached from the head, the human host 
is not freed from the infecting organism until the head has been passed, 
since it has the power of forming a new series of segments. The other 
is, that in examining stools for ova of tapeworms, it is important to 
examine for segments also, since ova are rarely found free in the stools 
but are contained in the intact segments. The ova of Dibothriocepha- 
lus latus, however, are almost constantly present in the feces. 

Taenia Saginata (Syns.: Taenia mediocanellata). — The length of the 
"beef tapeworm" ranges from 4 to 10 meters, and has even reached 
36 meters, the head is tiny, 1.5 to 2 mm. in diameter and cubical 
in shape. There are four suckers but no hooks, hence the name, 
"unarmed tapeworm." The rostellum is represented by a sucker-like 
organ. The segments are longer than they are broad. Each segment 



PLATE VII 




• 








/ 



C.L. Cummer. 



O lO 20 30 40 50 

I I I i I I I I I I 1 



Parasite Ova. 



^> 



a, Oxyuris vermicularis; b, Ascaris lumbricoides; c, Uncinaria americana; d, Hymenolepis 
la; e, Dibothriocephalus latus; /, Taenia saginata; g, Trichocephalus dispar. (All but / 
wn with camera lucida) . 



INTESTINAL PARASITES 



333 



has a single genital pore, which is laterally placed and alternates 
irregularly. The uterus shows a median trunk with twenty to thirty- 
five lateral branches, which ramify still further. There are two ovaries 
in each segment (Fig. 91). The approximately globular ova are about 
30 to 40m long and from 20 to 30^ broad. The inner egg-shell is usually 
intact and is surrounded by an embryonal shell which is thick and radi- 




Fig. 91. — Taenia saginata. 



6, head, much enlarged; c, ova, much 
(Simon.) 



ally striated. Larval development takes place in cattle. In infected 
cattle, encysted larvae (Cysticercus bovis) are found more frequently 
in the tongue and in the jaw-muscles than in other tissues. The 
cysticercus is rarely found in man. Infection is caused by eating 
uncooked or underdone meat. Distribution is extremely widespread. 
T. saginata is the species most frequently encountered in this country. 



334 



THE EXAMINATION OF THE FECES 



Taenia Solium (Syn. : Taenia dentata, Taenia huniana armata). — 
The average length of the "pork tapeworm" is from 2 to 3 meters 
though it may be longer. The globular head is very small, 0.6 to 1.0 
mm. in diameter. A short rostellum is armed with a double row of 
hooks, about 22 to 32 in number. There are four suckers, which are 
not as powerful as those of T. saginata. The segments increase in size 
gradually. Those shortly posterior to the head are almost square 
while those mature enough for detachment are longer than they are 
broad. The genital pores are on the side and alternate with con- 
siderable regularity. In contrast to T. saginata, the median trunk of 
the uterus has only seven to ten lateral branches on a side which in turn 
show tree-like branching. T. solium has three ovaries (Fig. 92) . The 






M 










1 2 3 4 

Fig. 92. — 1, head of Taenia solium; magnification, 50; 2, 3, mature and semi-mature 
segments, natural size; 4. two proglottides with uterus, twice magnified. (From Ziegler, 
after Leuckart.) 



eggs are quite similar to those of T. saginata and cannot be distinguished 
on microscopic examination. The fully developed adult form is found 
only in man. The larval stage (Cysticercus cellulosae) is found in the 
domestic pig commonly, and occasionally in some other animals, such 
as the dog, sheep, monkey, etc. The larval stage has been found also 
in man. T. solium, not often found on this continent, is much more 
frequent in Europe. 

Taenia Echinococcus. — In contrast to the two species of taenia just 
described, the T. echinococcus passes the adult stage of its development 
in the intestinal tract of a lower animal (dog) and the larval stage in 
man. It is described here not because the ova are found in the feces, 
since they are not, but simply to complete the description of the genus. 



INTESTINAL PARASITES 335 

Indeed, diagnosis is usually made only by finding the cysts on the 
operating table or at postmortem. 

The adult worm is one of the smallest of the tapeworms. There 
are only three or four segments and the total length is about 6 mm. 
The head shows four suckers and a double row of hooklets. The 
larval stage is found in hogs and sheep as well as in man. After the 
ova have been ingested, the embryo leaves its shell and bores through 
the walls of the alimentary tract, lodging in the liver or other organ 
where the cyst is formed. This is at first merely an envelope made up 
of a laminated wall enclosed granular fluid. Later it is seen that the 
wall has two layers. From the inner or germinal layer are developed 
brood capsules and scolices. In the enclosed brood capsules, the 




Fig. 93. — Segments of tapeworm: a, Taenia saginata; b, Bothriocephalus latus; 
c, Taenia solium. (Simon.) 

germinal layer is external. Scolices may be formed either on the inner 
or outer wall of the brood capsules. The original cyst is termed the 
mother cyst, the brood capsules forming daughter cysts. Ordinarily 
all of these contain scolices. The multiple cysts which are formed are 
termed hydatids. In examining cysts found at operation or autopsy, 
search should be made for fragments of laminated hyaline membrane 
and for hooklets. The latter are shed by the scolices. Man is infected 
by association with dogs. Hydatid disease is rather uncommon in this 
country but is frequent in Iceland, Central Europe, the Scandanavian 
peninsula, Australia, Paraguay and Argentine. 

Hymenolepis Nana (Syn. : Taenia nana).- — This is a small tapeworm, 
10 to 45 mm. in length (Fig. 94). The hooklets, about 24 to 30 in 



330 



THE EXAMINATION OF THE FECES 



number, are arranged in a single row. The segments are short and 
broad. The laterally placed genital openings, which cannot be seen 










Fig. 94.— Hymenolepis nana: 1, body; 2, natural size; 3, head; 4, hooklets; 5, eggs; 
6, egg magnified 600 times. (Mosler.) 

by ordinary means, are on the same side throughout. The distal 
segments are entirely rilled with eggs and disintegrate spontaneously. 



INTESTINAL PARASITES 



337 



It is stated by Stitt that after tsenicide treatment the entire worm is 
usually secured in an advanced stage of decomposition. 

The ova are oval or globular in appearance, 30 to 48^ across. There 
are two coats separated by intervening fluid substance. At either end 
of the inner coat is a projection from which proceed the filamentous 
bodies seen between the two coats. The intermediate stage of develop- 
ment is in the rat. The exact details are not known. The cystic 
stage (cercocyst) has been seen in man. It is thought that this may 
continue its development in the human host and give rise to a number 
of adult forms, thus accounting for the cases of heavy multiple infection. 
Cases are numerous in Sicily and in recent years many have been 
reported in the United States. In fact, this tapeworm is regarded by 
some as the most frequent in the United States. It may cause distinct 
symptoms, including loss of appetite and nervous disorders. 

Hymenolepis Diminuta (Syn. : Taenia diminuta). — The small tape- 
worm is common in rats and there are a limited number of cases on 
record of human infection. It measures 20 to 60 mm. in length. 
The segments are numerous, 600 to 2000 in number. The club-shaped 
head is small and is not armed with hooks. The round or oval eggs 
have a thick-yellowish outer coat showing radial striping, a thin, 
double, inner embryonal shell, and a middle granular layer between the 
two shells. The intermediate host is the rat flea. Human infection 
has been reported both on the American continent and in Europe. 

TABLE SHOWING CHIEF CHARACTERISTICS OF THE MORE COMMON 
TAPEWORMS. 



Dibothriocephalus Taenia solium, 
latus. 



Taenia saginata. I Hymenolepis i 



Length .... 


2 to 9 meters 


2 to 3 meters | 4 to 10 meters 


10 to 45 mm. 


Head 


Almond shaped, 2 


Globular, 0.6 to 1 ' Cubical, 1.5 to 2 


Globular, 0.25 to 3 




to 3 mm. long 


mm. diam. ; mm. diam. 


i mm. diam. 


Suckers .... 


Two grooves only 


Yes i Yes 


Yes. 


Hooks .... 


None 


2 rows I None 


20 to 24, in 2 rows. 


Number of segments 


3000 to 4000 


800 to 900 


More than 1000 


j Up to 200. 



Shape of segments 
Genital opening 

Uterus 

f Size 



[ Lid . . 
Intermediate host 



Usually broader 

than long 
Middle of segment 



Simple convolu- 
tions; near cen- 
, ter 
45 by 68-71 ju 

Thin; refractile 



Yes, at pole 
Fish 



Possible symptoms . Severe anemia 



Longer than broad Longer than broad| Broader than long. 

Alternately lateral, Irregularly alter- Lateral, all on same 
first on one side, nate | side, 

then on other 
Median tube with i 20 to 25 branches, 
lateral branch- \ breaking up into < 
ings 1 smaller tubes 

20 to 30 by 30 to Same as T. solium 30 to 48 M. 
40ju 

Same as T. solium 2 coats and inter- 
vening fluid sub- 
stance. 
None None. 

Beef , Rat. 



Thick, striated 



None 

Pig (dog, shee 
monkey, etc.) 



Nervous symptom. 



Dipylidium Caninum (Syn.: Taenia canina).— The adult worm is 
from 15 to 35 cm. long. The scolex has three or four rings of hooks. 
The mature segments are longer than they are wide, and are of reddish 
color. The genital pores lie symmetrically on the lateral borders. 

22 



338 THE EXAMINATION OF THE FECES 

The eggs are globular, 43 to 50/x, and have a thin internal shell, outside 
which is an albuminous coating, and external to both, a thin vitelline 
envelope. The parasite is common in dogs and is also found in cats. 
The intermediate host is the lice or fleas of the dog. Human infection 
occurs but is not especially common and is seen more frequently in 
young children than in adults. 

Dibothriocephalus Latus (Syn.: Bothriocephalus latus, Taenia lata, 
Bothriocephalic balticus, Bothriocephalus latissimus). — The "broad 
Russian tapeworm" has a length ranging from 2 to 9 meters or even 
longer. The head is almond shaped, and is about 2 to 3 mm. long. 
There are neither hooklets nor rostellum, the sucking apparatus con- 
sisting of two longitudinal grooves on either side of the head. The 
segments are extremely broad (Fig. 95). The uterus is rosette shaped 
and the genital pore is on the ventral side. The eggs are deposited in 
the intestine of the host and passed with the feces. They are rather 
large, 45 by about 70m, and brown. At one pole is a small lid. The 
embryo is ciliated, swims about in the water, and so enters the inter- 
mediate host. The intermediate hosts are various fresh-water fish. 
Infection takes place by eating raw or imperfectly cooked infected 






ESjZI I .'"ww'^*'' 



Fig. 95. — Bothriocephalus latus: a, b, twin segments. (Wilson.) 

fish, notably pike and perch. Human infection with the broad tape- 
worm is infrequently seen in this country, and when found is usually 
in an immigrant. It is found in Russia, Sweden, Poland, the coast 
districts of Germany, Switzerland, Turkestan and Japan, as well as 
in parts of France. It is of clinical importance on account of the 
resulting anemia which is often very severe and resembles pernicious 
anemia on clinical and laboratory grounds. 

Class Nematoda. — The nematodes (round worms) are bilaterally 
symmetrical filiform animals without limbs, usually round and elon- 
gated, and covered with a cuticle. The sexes are almost always sepa- 
rate. The male can usually be distinguished because of its smaller 
size and the spiral or incurved shape of the straight posterior extremity. 
In both sexes there is an excretory pore which is situated ventrally in 
the median line near the head. The genital opening of the female is 
near the midpoint in the median line while that of the male is near the 
anus. Most of the nematodes develop in damp earth or in water. 
Few of the nematodes are viviparous. 

Ascaris Lumbricoides. In the fresh condition the body is spindle- 
shaped and reddish-yellow or grayish-yellow in color (Fig. 96). The 



INTESTINAL PARASITES 



339 



oral papilla is surrounded with fine 
teeth. The male is from 15 to 25 cm. 
long and about 3 mm. in diameter. 
The posterior extremity is curved and 
the orifice of the cloaca is surrounded 
by numerous papillse. The testicular 
tube is visible through the body cov- 
ering and is much folded. The female 
is from 20 to 40 cm. long and about 
5 mm. *in diameter. Its posterior end 
is straight. The vulva is at the junc- 
tion of the anterior and median por- 
tions of the body. The ovaries are 
convoluted. The ova are elliptical in 
shape and are covered with a thick, 
shaggy coat, so that when the egg is 
seen under the microscope it appears 
to be covered with knobs or protuber- 
ances. Inside of this is a thick, trans- 
parent shell. The egg-cell itself is 
unsegmented and the nucleus cannot 
be seen because of the coarse granules. 
When the ova are found in the feces, 
they show a brown coloration of the 
outer coat due to staining with bile 
pigment. 

A . lumbricoides is almost universally 
found, but is more common in warmer 
climates. It usually infects children. 
Only a few worms may be present, 
though large numbers have been re- 
ported. Normally, the habitat is the 
small intestine, though the worms may 
migrate from here to the stomach (and 
so be vomited up), and to other parts 
of the body, where they may be the 
cause of "worm-abscesses." They have 
even gained access to the genito-urin- 
ary tract and have been passed in 
the urine. The eggs develop in moist 
soil but do not leave the shell until 
taken into the intestinal tract. Infec- 
tion is by swallowing infected soil. 

Belascaris Cati (Syns. : Belascaris 
mystax; Ascaris mystax).— This be- 
longs to the family of Ascaridse. The 
worm is much smaller than the Ascaris 
lumbricoides, the male being 3 to 6 
cm. long and the female 4 to 10 cm. 
long. From the anterior end of the 




Fig. 96. — Ascaris lumbricoides: A, 
female; B, male; C, egg. At a, the 
female genital opening; b, the en- 
larged cephalic extremity, with its 
three lips; c, the male spicules. 
(After Perlo, from Ziegler.) 



340 



THE EXAMINATION OF THE FECES 



parasites are wing-like, bilateral projections, which give the parasite 
an arrow-like appearance. The eggs are 65 to 7-V in diameter with 
finely honey-combed surfaces. — The host is the domestic cat and dog, 
rarely man. 

Oxyuris Vermicularis. — These worms are popularly known as "pin- 
worms," " seat-worms," or " thread-worms" (Figs. 97, 98 and 99). They 





Fig. 98. — 1, Oxyuris vermicularis: a, 
male; b, female; natural size. 2, magni- 
fied. (Simon.) 




Fig. 97. — Oxyuris vermicularis: 
a, sexually mature female; b, female 
filled with eggs; c, male. Magnifi- 
cation, 10. (After Heller, from 
Ziegler.) 



Fig. 99. — Eggs of Oxyuris vermicularis 
in various stages of development: a, b, c, 
division of the yolk; d, tadpole-like em- 
bryo; e, worm-shaped embryo. Magnifi- 
cation, 250. (After Zenker and Heller, 
from Ziegler.) 



are white and small, the males measuring 3 to 5 mm. in length and the 
female 10 mm. in length. The tail of the males is incurved. The 
striated cuticle at the anterior end forms a bulbous-like projection. 
The eggs are symmetrical and oval, measuring from 50 to 55/x by 16 to 
25/x- The shell has a double contour. When the ova are deposited, 
the embryo within them has already developed. 



INTESTINAL PARASITES 341 

After ingestion of material containing oxyuris eggs, the adults 
develop in the intestine where copulation takes place. The male then 
dies. When the uterus of the female begins to fill with eggs, they leave 
the cecum and go to the rectum. Here the eggs are laid, inside and 
outside of the anus. This usually occurs at night and causes intolerable 
itching. The worms which are hatched may wander to adjoining parts 
of the body and may be carried under the patient's finger nails to 
distant parts. An intermediate host is not required for development, 
so that reinfection of the patient is possible by carrying the eggs under 
the nails to the mouth. Diagnosis is best made by the examination 
of the stools after the administration of calomel and salts for the thread- 
like female adults, which are visible to the naked eye. The worker 
should be careful not to soil the hands with fecal material, since infec- 
tion is possible. 




Fig. 100. — Ankylostoma duodenale, male and female. Natural size. (Mosler.) 




Dorsolateral ray 
Lateral ray 

Subventral 
ray 

Ventrolateral 

ray 



Ventral ray 



J 



w 



Fig. 101. — A, caudal bursa and tail of male Uncinaria duodenalis; B, caudal bursa 
and tail of male Uncinaria americana. Drawn to scale to show difference in size. (After 
Allen J. Smith.) 

Anchylostoma Duoden,ale.— This is commonly known as the Old World 
species of hook-worm (Figs. 100, 101 and 102) . The worms of both sexes 
have a mouth capsule which is armed with teeth. There are two pairs 
on the ventral surface and two protuberances which are rea ly rounded 
edges of a gap in the edge of the capsule. The cutting apparatus also 
includes a pair of lancets. The body shows a marked lateral torsion 
and the head is curved dorsally somewhat. The male measures about 



342 



THE EXAMINATION OF THE FECES 




Fig. 102. — Male Ankylostoma duodenale: a, head; b, esophagus; c, gut; d, anal glands, 
e, cervical glands;/, skin: g, muscular layer; h, excretory pore; i, trilobed bursa; A;, ribs' 
of bursa; I, seminal duct; m, vesicula seminalis; n, ductus ejaculatorius; o, its groove- 
p, penis; q, penile sheath. Magnification, 20. (After Schulthess, from Ziegler.) 



INTESTINAL PARASITES 343 

9 mm. long by 0.45 mm. thick and the female 12 mm. long by 0.6 mm. 
thick. The male may be distinguished by a wide umbrella-like 
expansion of the posterior end (copulatory bursa) while the tail of the 
female is pointed. The vaginal opening of the female opens posterior 
to the median line. The eggs which are oval with rather broad poles, 
measure about 34 to 38/x by 56 to 61/x- The contour is single. Between 
the shell and the granular mass of the ovum is a very striking clear 
space, which renders the recognition of the egg quite simple. The 
ovum often shows segmentation into four masses. Embryos are not 
seen within the eggs in freshly passed stools. The parasites live in the 
jejunum, less frequently in the duodenum. They attach themselves 
to the mucosa, and so cause loss of blood. Larvae enter the body 
either by the mouth or burrowing through the skin. When the skin 
is penetrated, there is redness and burning at the point of entry and 
later swelling of the connective tissue. Through the lymphatics or 
the blood stream the parasites ultimately reach the intestinal tract. 
After the eggs have been passed in the stools, the embryos hatch. 
The embryo (larva) develops under suitable conditions of temperature 
(best between 25 and 30° C.) and in the presence of oxygen. Man 
only is infected. An intermediate host is not required. The distribu- 
tion is fairly wide, including Africa, Egypt, Japan, China, Southern 
Europe, portions of South America and the Philippines. 

The symptoms produced may be very marked, including lassitude, 
pallor, underdevelopment, loss of weight, anemia of the secondary 
type of more or less marked degree, the hemoglobin falling as low as 
40 or 50 per cent. A differential count usually shows an increase in 
the percentage of the eosinophiles. 

Necator Americanus.— The New World hookworm, first noted in the 
United States by A. J. Smith and first described by Stiles, shows certain 
marked points of differentiation from the Old World species. The 
buccal capsule is small and there are no ventral teeth as in A. duodenale 
but instead cutting plates, with a dorsal cone which projects into the 
buccal cavity. Four buccal lancets are seen. The head is bent sharply 
dorsalwards. The male is 8 'mm. long and the female 10 mm. long. 
The opening of the vulva in the female is in front of the median line. 
The eggs are somewhat more pointed at the poles than the eggs of 
A. duodenale, and measure about 36 by 64 to 72/jl. The infection 
is very prevalent in the southern portion of the United States and in 
Porto Rico. 

Strongyloides Intestinalis.— The life-history of this parasite includes 
two generations, a parasitical or intestinal form, and a free-living form. 
The former is sometimes referred to as S. intestinalis and the latter as 
S. stercoralis. The parasitic generation is represented only by the 
female. In the opinion of most workers reproduction is by partheno- 
genesis, although some believe that the parasite is hermaphroditic 
and that the testes degenerate after functioning. The parasite is 
colorless and in situ can scarcely be seen even with a hand lens, so that 



344 



THE EXAMINATION OF THE FECES 



it is necessary to scrape off the mucosa and to examine the scrapings 
microscopically. The worm is only about 2.2 mm. in length and from 
34 to 70ju in breadth (Fig. 103). The mouth is surrounded by four 
lips. The anus opens shortly in front of the posterior extremity. The 
vulva is situated at the junction of the middle and posterior thirds of 
the body. The eggs resemble the eggs of Anchylostoma duodenalis in 
appearance. They are about 30 to 34^ across and from 50 to 54^ long 
and lie in a chain. The eggs are laid in the mucosa of the host and are 




Fig. 103. — A, egg of Strongyloides intestinalis (parasitic mother worm); B, rhabditi- 
form embryo; C, filariaform embryo, derived by direct transformation from arhabiditi- 
form embryo. (Thayer.) 



rarely seen in the stools except after active purgation. The stools, 
however, may show actively motile embryos which have pointed tails 
and at first measure about 250/x in length, growing to a length of 500/x. 
When the temperature is low, the rhabditiform embryos develop into 
filariaform embryos, which are infective when ingested. At a higher 
temperature the parasites become sexually mature, copulate, and form 
the free-living generation. 

The free-living form has a smooth cylindrical body, pointed at the 



INTESTINAL PARASITES 345 

posterior end. The mouthTshows four indistinct lips. The males 
measure 0.035 mm. in breadth and 0.7 mm. in length. Their posterior 
end is curved. The females have a straight tail and are 0.05 mm. broad 
by 1 mm. long. After the eggs have emerged from the shell, rhabditi- 
form larvae develop with all the characteristics of the parents. When 
sufficiently grown, they moult and assume the characteristics of the 
grandparents (parasitic generation). When ingested, they become 
parasitic; otherwise death takes place. 

The parasite was first discovered in so-called Cochin China diarrhea. 
It is found in Brazil, Africa, and Europe, and cases have been reported 
in the United States. Infection of man may take place not only by 
direct entry into the stomach but through the skin. 

Trichinella Spiralis (Syn. : Trichina spiralis) . — The adult male 
worm measures about 0.04 mm. in diameter by 1.4 to 1.6 mm. in 
length. The cloaca is at the posterior end and lies between two caudal 
appendages which serve to hold the female during copulation. The 
cloaca is evertible and forms a penis. The female is 0.06 mm. in diame- 
ter and from 3 to 4 mm. long. The anus is terminal and gives off 
embryos (not ova) from the vulva, which is near the anterior end. 

In the adult stage, the parasite inhabits the small intestine of man, 
pig, rat, and wild boar. The adult males die shortly after copulation. 
The young remain in the body of the host, are deposited by the female 
in the lymph spaces and carried by the lymph stream to the heart. 
In the early stage of infection, they may be found in the blood by 
centrifugalizing a small quantity which has been laked with ten times 
its volume of dilute acetic acid. Though they are distributed to 
various parts of the body, the larvae live only in the striated muscle, 
where they encyst themselves. The cysts, which are surrounded by a 
chitinous capsule, measure about 250 by 450//. The larva are seen 
curled up in the cysts. They measure about 6ju in diameter by 90 to 
100/i in length. They may be found in any of the striated muscles, 
especially in the tongue muscles and the diaphragm. Diagnosis may 
be made by excision of a bit of the deltoid or biceps muscles near the 
insertion and by teasing out the tissue under the microscope to demon- 
strate the encysted larvse. A marked eosinophilia is present, some- 
times running as high as 50 per cent. 

Man is infected by eating infected and insufficiently cooked pork. 
Pigs are infected by eating rats, who in turn are infected by eating- 
pork about slaughter houses. Since they are cannabalistic, rats 
continue the infection indefinitely. During the period of invasion, 
there are marked gastro-intestinal symptoms, vomiting and diarrhea. 
There may be slight edema. In the second week, myositis is promi- 
nent. Mastication, speech, and respiration may be difficult. Eosino- 
philia is found. The temperature may be elevated to 104 or 105° F. 
In the third week, encystment takes place. Edema of the face is 
pronounced. There may be delirium and somnolence. Death may 
result or the milder cases may progress to recovery. 



346 



THE EXAMINATION OF THE FECES 



Trichuris Trichiura (Syn.: Trichocephalus trichiurus, Trichocephalus 
dispar).— This parasite, commonly known as the "whipworm," lives 
in the cecum of man, occasionally in the appendix, colon, or even 
small intestine (Figs. 104 and 105). The male measures 40 to 




Fig. 104.— Trichocephalus dispar. a, male; b, female. (Mosler.) 




Fi<;. 10o.— Trichocephalus dispar. A, male; B, posterior extremity of female- C 
ovum, a head; b, cephalic extremity of body with esophagus; c, stomach- d gut'- 
e, cloaca;/ seminal canal; g, penis; /, bell-shaped penile sheath, with tip of penis- m 
gut of female; n, anus; o, uterus; p, vaginal cleft. Magnification, 10. (After Kiichen- 
meister and Ziirn, from Ziegler.) 

45/i in length, the female 45 to 50 M . In both sexes, the anterior 
portion of the worm is s'ender and whip-like. The ova are striking 
and characteristic, being barrel-shaped and dark-brown in color, with 
a perforation at each pole which is closed with a plug of light color. 



PRESERVATION AND STAINING OF FLUKES AND CESTODES 347 

They measure about 23yu in breadth and from 50 to 54/x in length. The 
eggs develop in water or moist soil. Infection in man is common. 
There as considerable question as to whether any symptoms result. 

PRESERVATION AND STAINING OF FLUKES AND CESTODES. 

Fixation of Flukes.— The flukes should be placed in a small bottle 
or test-tube about a quarter full of normal saline solution. The 
contents are shaken well to extend the worm and an equal bulk of a 
saturated solution of mercuric chloride (10 gm. to 160 cc. of water) 
is added. This is shaken vigorously for a few moments, when the 
worm is transferred to 70 per cent, alcohol. 

For large flukes, the worm should be flattened out between two 
glass slides held together by rubber bands, and fixed by immersion in 
a saturated solution of mercuric chloride. 

Staining may be accomplished after fixation. The mercuric chloride 
should be removed with tincture of iodine. Then the parasite is 
allowed to stand overnight in a dilute solution of carmine. Differentia- 
tion may be accomplished by allowing the preparation to remain in acid 
alcohol from one to twenty-four hours. (Acid alcohol may be pre- 
pared for this purpose by adding 5 drops of HC1 to 100 cc. of 70 per 
cent, alcohol.) The specimen should then be dehydrated by placing it 
in increasing strengths of alcohol, when it may be cleared in phenol 
(phenol, 4 parts, water 6 parts). It may then be transferred to 95 
per cent, alcohol, next to xylol, and finally to balsam; or it may be 
mounted in glycerine, according to the method for ova given below. 

Fixation of Cestodes. — The worm is placed in a saturated so'ution of 
mercuric chloride to which acetic acid has been added to the extent of 
1 per cent. Plenty of fixation should be used. The fixative should be 
warmed to 70 or 80° C. and allowed to act for fifteen minutes. The 
worm may then be transferred to 70 per cent, alcohol. The mercuric 
chloride should be removed by treatment with tincture of iodine. 
Staining, dehydration, and mounting may be accomplished as directed 
for the flukes. 

Preservation of Ova in Feces. — Seventy per cent, alcohol is heated in 
a beaker to a temperature of about 70° C. The feces are added, about 
one part of feces for every 9 parts of alcohol. The mixture is stirred 
and then allowed to settle. The sediment may be transferred to a 
bottle of fresh 70 per cent, alcohol. 

The ova may be kept in a glycerine jelly. Three strengths of gly- 
cerine are prepared in 70 per cent, alcohol, 5, 10 and 20 per cent. The 
alcohol is poured off the sedimented feces, and is replaced with 5 per 
cent, glycerine. After standing for an hour or two, this is in turn 
poured off and replaced with 10 per cent, glycerine, which is in turn 
replaced with 20 per cent, glycerine. The latter is allowed to stand, 
exposed to the air but protected from dust, so that the water and alcohol 
will evaporate. A few drops of glycerine are added from time to time 
so that the ova are finally in pure glycerine. 



CHAPTER V. 
THE EXAMINATION OF SPUTUM. 

Precautions Against Infection. — The specimen should be handled with 
care. Spread a newspaper on the work-table and perform all steps 
on it. When through, fold the paper up and burn it or have it auto- 
claved. A basin containing 1 : 20 phenol solution should be at hand. 
When the examination is completed, put into it corks, extra slides, 
bottle, Petri dishes, or any other glass-ware which has come into 
contact with the sputum. This should then be sterilized either in the 
autoclave or by boiling. If particles of sputum are dropped on bench 
or floor, flood with 1 : 20 phenol or 1 : 1000 mercuric chloride at once. 

Obtaining the Specimen. — When a patient is asked to furnish a speci- 
men of sputum he should be told that the sputum desired is that which 
he coughs from "deep down" (from the lungs) and not that which comes 
from the mouth, nose or throat. In office or dispensary practice the 
patient should be furnished with a clean, dry, wide-mouth bottle. 

Small children usually swallow sputum. Sputum may be obtained 
from them by irritating the pharynx with a cotton-wrapped swab or 
with the finger, wrapped with a piece of sterile gauze. The sputum 
raised by the resulting cough can be caught on the swab or gauze. 
Another method is to obtain the stomach contents mixed with sputum 
by passing a small catheter. 

Method of Procedure. — The sputum should be subjected to careful 
macroscopic as well as microscopic examination. The following 
serves as an outline of the points which should be covered on a routine 
examination : 

(a) Macroscopic Examination. — Amount; consistency; odor; color; 
presence of fibrinous casts, Curschmann's spirals, Dittrich's plugs, bits 
of lung tissue or cartilaginous rings, parasites, concretions or foreign 
bodies, ray fungus particles. 

(b) Microscopic Examination— Unstained Specimen.— Leukocytes; 
epithelial cells; red blood cells; heart failure cells; elastic fibers; 
Curschmann's spirals ; moulds ; parasites ; crystals. Stained Spec i m ens.— 
Stain with Gram's stain; stain for tubercle bacillus; note the types 
of epithelial cells and leukocytes. 

(c) Special Examinations.— -When indicated, specimens may be 
stained with special stains to demonstrate other microorganisms and 
eosinophilic leukocytes. 

ORDER OF PROCEDURE. 

The gross appearance of the sputum should be noted. Prepare 
slides and stain for tubercle bacilli. Make a slide by the "impression 



GROSS APPEARANCES OF THE SPUTUM 349 

method" and stain with Gram's stain. While decolorization is going 
on, examine microscopically a particle of unstained sputum. Special 
stains, as Wright's stain to demonstrate eosinophilia, stains for influ- 
enza bacillus and for capsules, etc., should be made when the clinical 
or the preliminary findings with Gram's stain point to their desirability. 



GROSS APPEARANCES OF THE SPUTUM. 

While the gross characteristics of the sputum should be studied and 
noted carefully when a report is made, the laboratory worker should 
not allow himself to be biased by the appearances, nor should' he allow 
the apparently normal gross appearance of the sputum to deter him 
from making a careful microscopic bacteriological examination. 

Description.— In describing the sputum., use is usually made of the 
terms serous, mucous, mucopurulent, or bloody. These are self- 
explanatory. The macroscopic description should be completed with 
notation of the following characteristics: 

Amount.— This can usually be observed only in those who are con- 
fined to their beds. The statements of the ambulatory patients regard- 
ing the amount of the expectoration are often unintentionally mis- 
leading. The amount is greatly increased in edema of the lungs, in 
bronchorrhea, acute bronchitis (especially with cavity formation), 
in pulmonary tuberculosis with cavity formation, in abscess or gan- 
grene of the lungs, and after the perforation of an empyema into the 
pulmonary tissue. 

Consistency.— The sputum is of thin, watery consistence in edema of 
the lungs and occasionally in abscesses, gangrene, and large tuberculous 
cavities. It is especially thick and tenacious in bronchial asthma, the 
earlier stages of pneumonia, and occasionally in catarrhal conditions. 

Color. — Pale yellow or green sputum is occasionally seen in acute 
bronchitis. The sputum in pulmonary edema has a bright pink tinge. 
Red coloration is usually due to the presence of blood. A bright-red 
color is seen when a small vessel has been ruptured and occasionally in 
pulmonary infarction. 

A brownish-red color is due to changed blood and may be seen in 
pulmonary gangrene. Dark-brown, "prune-juice" colored sputum is 
expectorated in certain stages Of pneumonia. A bright-green color, 
due to the presence of bile pigment, is sometimes seen after the rupture 
of a liver abscess into the lung. A green color is also seen at times in 
pneumonia, especially during slow resolution where the blood pigment 
is changed to hematoidin and this is oxidized to biliverdin. Black 
sputum is seen in coal-handlers and in those who dwell in regions 
where much soft coal is burned. 

Odor.— Ordinarily the sputum is odorless, though the expectoration 
from large bronchiectatic cavities and from gangrene of the lung has a 
particularly offensive odor. 



350 



THE EXAMINATION OF SPUTUM 



Fibrinous Casts. — Casts of the bronchi, representing a portion of the 
bronchial tree, may be quite extensive in size (Fig. 106). They may 




Fig. 106. — Expectorated cast from a case of fibrinous bronchitis. Three-fourths natural 
size. Drawn from fresh specimen. (After Bettmann.) 






Fig. 107. — A Curschmann spiral from a case of true bronchial asthma (enlarged). 
(Simon.) 



be seen in fibrinous bronchitis, in pneumonia during the stage of con- 
solidation and in diphtheria. 



MICROSCOPIC EXAMINATION 351 

Curschmaim's Spirals. — These small, spiral bodies, which are visible 
macroscopically, may occur in two forms, either as spirally-twisted 
strands of mucus in which are enclosed leukocytes (particularly 
eosinophiles) and Charcot-Leyden crystals, or as mucus twisted around 
a central fiber (Fig. 107). They are seen in cases of bronchial asthma, 
particularly at the end of the paroxysm, and occasionally in acute 
bronchitis, acute lobar pneumonia, and chronic pulmonary tuberculosis. 

Dittrich's Plugs. — Dittrich's plugs are bodies varying in size from that 
of a millet seed to that of a bean. They are of yellowish-white color 
and when crushed give rise to a most disagreeable odor. Micro- 
scopically they are found to contain cell detritus, fatty acid crystals, 
fat drops, and bacteria. They are from the bronchi and are quite 
similar in appearance to the plugs which are squeezed from the tonsillar 
crypts. 

Bits of Necrotic Tissue. — Bits of necrotic tissue are seen occasion- 
ally in cases of gangrene and abscess, and more rarely in pulmonary 
tuberculosis. Often they are so small that microscopic examination is 
necessary to determine their true nature. They may be portions of 
pulmonary tissue or fragments of cartilaginous rings from the cartilages 
of the traches, bronchi, or larynx. 

Concretions.— In most cases, these are calcified tuberculous material. 

Parasites.— With echinococcus infection of the lungs, cysts may be 
expectorated and hooklets have been identified in the sputum. Parti- 
cles of ray fungus may be seen in the form of sulphur granules. 

MICROSCOPIC EXAMINATION. 

Unstained Specimen.— Much information may be gained merely 
from the examination of an unstained specimen. In fact, the "so- 
called heart-failure" cells or elastic fibers would be overlooked in the 
examination of the specimens stained by the usual methods. While 
making this examination, the number of the leukocytes, number and 
type of epithelial cells, and presence of red blood cells should be noted. 
After completing the examination, care should be taken to sterilize 
the slide. 

The field should be darkened by dropping the Abbe condenser and by 
partially closing the diaphragm just beneath the stage. The material 
should be placed on an object slide, and covered with a cover-slip. 

"Heart Failure Cells" (Herzfehlerzellen).— The alveolar endothelial 
cells are four or five times the size of a leukocyte, oval in outline, with 
granular cytoplasm and a vesicular, oval nucleus. They are found in 
greater or less number in all sputa. In cases of chronic passive pul- 
monary congestion due to cardiac decompensation, they may become 
partially filled with hemoglobin pigments, which may be in the form of 
hematoidin crystals, or amorphous masses or scales of hemosiderin. 
When the pigment is hemosiderin, it will give an iron reaction when 
treated with a dilute solution 5 per cent, potassium ferrocyanide and a 



352 THE EXAMINATION OF SPUTUM 

dilute 5 per cent, solution of hydrochloric acid. This reaction may be 
carried out on the slide by running the reagents under the cover-slip, 
watching the results under the microscope. The granules turn 
Prussian-blue. Recognition of these cells may have some importance 
in completing the picture of bronchitis of cardiac origin. Care should 
be taken not to mistake for "heart failure cells," alveolar cells which 
merely contain black particles of carbon. 

Curschmann's Spirals.— The macroscopic appearance has been 
described. On microscopic examination they will be found to consist 
of spirals in which are enmeshed eosinophilic cells and Charcot-Leyden 
crystals. Examinations should be made with both low power and high 
power objectives. 

Elastic Fibers. — Elastic fibers may be found in gangrene and abscess 
of the lung and in pulmonary tuberculosis. Their recognition may be 
of considerable importance in the diagnosis of early pulmonary tubercu- 
losis. They may be seen when the sputum is pressed between two 
glass plates in the Sir Andrew Clark method. In making the search, 
a black background should be employed. Suspicious bodies may be 
picked up with a teazing needle, placed on a slide and covered with a 
cover-slip. The fibers will be seen arranged as though about the 
alveolar structure. 

If it is not possible to find them in this way, the sputum may be 
digested with sodium hydrate which serves to dissolve everything 
except the more resistant elastic tissue. A portion of the sputum 
is boiled with approximately an equal quantity of 10 per cent, sodium 
or potassium hydrate. After the mixture has been allowed to stand 
overnight, it is centrifugalized and the sediment placed on a slide and 
examined. If desired, the fibers may be stained with orcein. 1 The 
supernatant fluid is poured off the sediment, and distilled w<ater is 
added in order to free the sediment from alkali. The tube is again 
centrifugalized and the water is poured off. A few drops of the orcein 
stain are added, when hydrochloric acid is dropped in until a violet 
color appears. The tube is placed in boiling water for about five 
minutes, when the contents are decolorized with acid alcohol. 2 

Fibers from food material can be distinguished only by the absence 
of alveolar formation in their arrangement. 

Crystals.— Under occasional circumstances, various forms of crystals 
may be seen in sputum. The appearance of most of the crystals has 
been described in the chapters on either Urine or Feces. 

Charcot-Leyden crystals are colorless, octahedral in form, dissolving in 
water and acetic acid and staining with eosin (Fig. 108). They are 
found in the sputum of cases of bronchial asthma and are often en- 
meshed in Curschmann's spirals. Lencin and tyrosin arise from the 

1 Orcein stain is prepared by dissolving a gram of orcein in 80 cc. of 95 per cent, 
alcohol, and 35 cc. of distilled water, adding 40 drops of strong hydrochloric acid. 

2 Acid alcohol for this is prepared by adding 1 cc. of strong hydrochloric acid to 
200 cc. of 80 per cent, alcohol. 



MICROSCOPIC EXAMINATION 353 

decomposition of protein. They are seen in sputum after the rupture 
of an empyema into the lung and occasionally after the perforation of 
a liver abscess into the lung. Fatty acid crystals are seen in gangrene 
of the lungs and occasionally in chronic phthisis. Cholesterol crystals 
are found in the sputum of cases with chronic lung abscesses and 
where there are cavities as in chronic tuberculosis. Hematoidin 
crystals are in the form of rhomboids from whose corners project 
needles or curved filaments. They are bright yellow in color and 
dissolve in chloroform. They occur in the sputum rarely, appearing 
only when extravasated blood has remained in the pulmonary alveoli 
for a long time. Hypomycetes (Molds). Various forms of molds 
are often found in the sputum as a result of contamination. Certain 
varieties may be pathogenic. There are hyphse (jointed rods) and 
spores. The rods are often branched and are frequently arranged in 
the form of a network. The columella is the rounded termination 
of an aerial hypha, which projects upward and carries the fruiting 
organism bearing the conidia or spores. 



B®iJ 




O 



Fig. 108.— Charcot-Leyden crystals. (Scheube.) , 

An organism, which they have termed Monilia, has been described 
by Boggs and Pincoffs in a fatal case with abscesses in breast and axilla 
communicating with a cavity between the two layers of the pleura. 
The sputum was thin, gruel-like, odorless, and faintly tinged with 
blood and contained small gray masses which showed mycelial 
threads and yeast-like cells when examined microscopically. The 
organism could not be distinguished from that described by Ashford 
in tropical sprue. Both hyphee and sporulation forms were Gram- 
negative. Growth on glucose agar was luxuriant. 

Blastomycetes (Yeasts).— The blastomycetes reproduce in the tissues 
by budding or by sporulation only. They may be found in the 
sputum in pulmonary blastomycosis, (Fig. 109) and are apparently 
identical with the organisms found in pus and tissues in subcutaneous 
blastomycosis. The infection is systemic, as shown by the studies of 
Montgomery and Ormsby and Stober and others. They are seen in 
the sputum as round or oval bodies with a highly refractile, double 
23 



354 



THE EXAM1XATI0X OF SPUTUM 



contour. They are Gram-positive, stain readily with ordinary stains, 
and may be recognized microscopically even in unstained specimens. 
The organism may be cultivated on ordinary media. Growth is seen in 
from two to fourteen days. In cultures kept at room temperature, aerial 



^'3 



r 3 



..' 







Fig. 109. — Blastomycetes. Smear from sputum mounted in 1 per cent, potassium 
hydrate solution, showing circular and budding organisms. X 1200. (Eisendrath and 
Ormsby.) 




Fig. 110.— Crushed 



mle from fresh sputum. Unstained. X 750. 
(Lord.) 



MICROSCOPIC EXAMINATION 355 

hypha? are formed and microscopic examination shows finely branching 
mycelium with a few small, spore-like bodies, while cultures kept at 
37° C. show a moist growth and budding forms. 

Actinomycosis.— The characteristic finding in the sputum is the 
"sulphur granules." Macroscopic recognition is important. When 
one of the "granules" is pressed out on a slide beneath a cover-slip, it 
will be seen to consist of a net-work of fine threads, at the ends of which 
are bulbous-shaped granules (Fig. 110). The central portion may be 
stained with Gram's stain. The bulbous ends are degenerate struct- 
ures and stain with eosin though not with Gram's stain. The organ- 
ism may be cultivated in deep glucose agar stabs since it is essentially 
an anaerobe. 

Parasites.— Attention has been called to the possibility of finding the 
hooklets from echinococcus cysts in the sputum. These maybe identi- 
fied microscopically. Amebos may be found from amebic liver abscesses 
which have ruptured into the lung. In looking for them, the technic 
described elsewhere (see page 325) should be followed. Filarial 
embryos and the ova of Paragonimus westermannii are seen in the 
sputum. These ova, which are constantly present in the expectoration 
of infected individuals, are brownish-yellow and about 49 to 81/x in 
diameter. The lung- fluke itself may be expectorated. It is plump, 
oval in shape, and faint reddish-brown in color. It is 3.5 mm. thick, 
4 to 6 mm. wide, and 7.5 to 12 mm. long. There are two suckers, 
the oral one below the anterior termination, the ventral one in front of 
the middle of the body. The surface is covered with spear-shaped 
spines. The branched ovary is on one side, the uterus is on the 
opposite side, and testicles are posterior. 

Man and dog are hosts for the adult parasite. The flukes are found 
in cysts in the lungs. The cyst walls are formed of thickened connec- 
tive tissue. The larval stage (cercaria) is found in two intermediate 
hosts, molluscs and fresh-water crabs. When ingested by man, it is 
thought that the fluke goes through the intestinal wall, perforates the 
diaphragm, invades the pleural cavity, penetrates the lung tissue and 
there becomes encysted. The symptoms are those associated with 
bronchitis and peribronchitis, cough and hemoptysis, so the infection 
has been called endemic hemoptysis. The distribution of the lung- 
fluke is in Japan, China, and Korea. 

Examination of Stained Specimens.— Impression Method of Making 
Preparations.— In staining for organisms other than tubercle bacilli 
and especially when a study of the cell morphology is desired, the 
"impression method" of making slides should be followed. A drop of 
mucopurulent material is taken up on a platinum wire' and touched 
to the surface of the slide in a number of different places. It should 
not be smeared over the surface lest the cells be broken up. 

For general examination, a slide should be stained with Gram's 
stain and one with Loeffler's methylene blue or with toluidin blue. 
When it is desired to determine the presence of eosinophiles, as in 



356 THE EXAMINATION OF SPUTUM 

bronchial asthma, a slide made by the impression method should be 
stained with Wright's stain. 

Preparing Slides for the Search for Tubercle Bacilli.— The best method 
to employ in hunting for tubercle bacilli is that of Sir Andrew Clark. 
Two pieces of glass (preferably plate-glass) are necessary. One of 
these should be about 14 to 18 inches square and the other about 6 
inches square. A portion of the sputum is placed on the upper surface 
of the larger piece of glass, and the small piece of glass is placed over the 
sputum, just as a specimen on an object slide is covered with a cover- 
glass. The upper piece of glass is pressed down rather firmly and is 
moved along gradually, while the lower piece remains stationary. 
The specimen is now spread out in an extremely thin layer. Close 
watch is kept for the tiny cheesy bodies, which are picked up with a 
teasing needle and placed on an object slide as they emerge at the edge 
of the smaller piece of glass. After four or five have been obtained, a 
smear is made on an object slide by placing over it a second slide, which 
is pressed down and then pulled away so that a film is made on both 
slides. 

The only objection to this method is the inconvenience of sterilizing 
the rather unwieldly pieces of glass, especially when many specimens 
are to be examined. It is more convenient to pour the specimen into 
Petri dishes. With either method, a dark background should be used. 
A piece of black paper may be placed under the glass plate or Petri 
dish. The "rice bodies" or cheesy particles should be looked for with 
great care. Simple mucus or pure blood or pus should be avoided. 
Particles of food are often confusing. A teasing needle will be found 
better suited for picking up the cheesy bits than a platinum loop. 

Staining for Tubercle Bacilli.— Fixation.— Dry the slides by holding 
them high over a low flame. Then fix them by passing them quickly 
through the flame three times, holding the slide with the smeared side 
down. Care should be taken to avoid overheating or scorching the 
smears. An unfixed slide may bear infectious material and should be 
handled accordingly. If it be not stained, it should be passed through 
the flame several times and thrown in a receptacle containing an anti- 
septic solution. 

Ziehl-Neelsen Method.— After covering the slide with Ziehl-Neelsen 
carbol-fuchsin solution, hold it over a low flame to steam. The 
stain must not be allowed to boil or to dry around the edges, since 
;i crystalline deposit is formed which makes a deceptive preparation. 
Steaming should be continued for at least three minutes, adding fresh 
stain to replace that lost by evaporation. Many specimens may be 
stained at the same time by placing them in a beaker or other vessel 
which is filled with steaming carbol-fuchsin, marking the slides for 
identification. 

The stain is poured oil' and the film decolorized by immersing in acid 
alcohol (1 part of strong hydrochloric acid in 99 parts of 85 per cent, 
alcohol) for a few seconds until almost colorless, when it is placed in 



PLATE VIII 

1 






Photograph of Human Type of Tubercle Bacilli from Sputum. 

laeiJli in red, rest of specimen blue. X tOOO diam. (Frankel and Pfeiffer). 



MICROSCOPIC EXAMINATION 357 

05 per cent, alcohol until it is evident that no more color is removed. 
This will require from one to five minutes. The film is counterstained 
with Loeffler's methylene blue for one minute, washed with water, 
and dried, and examined with the oil-immersion lens. A mechanical 
stage is needed for careful search. 

The tubercle bacilli appear as bright red, granular or dotted rods 
since they are "acid-fast" (resisting the decolorization of the acid), 
while other bacteria, leukocytes, and epithelial cells are blue (Plate VIII) . 
This method of staining is so far superior to Gabbett's method that the 
technic for the latter will not be given here. While Gabbett's method 
is somewhat more rapid the results are not as reliable and the appear- 
ance of the slide stained with Gabbett's fluid is often far from satis- 
factory. 

Pappenheim's Method.— The slides are stained with carbol-fuchsin 
as with the previous method. The stain should be poured off without 
washing and the slide flooded with the Pappenheim's decolorizing fluid. 
This is poured off slowly, and the slide covered with fresh fluid, which in 
turn is drained. The process is repeated four or five times. While 
smegma bacilli may retain the stain after decolorization with the 
mineral acids and alcohol, they are decolorized by rosolic acid. The 
method is particularly suited for the examination of urine, where the 
possibility of contamination with smegma is much greater than in 
examining sputum. 

Much's Granules.- — Much described granules which are Gram-positive 
but not acid-fast. They are regarded as tubercle bacilli because when 
material containing them has been injected into susceptible animals, 
tuberculosis has resulted. Since they are found in inactive or quies- 
cent cases the claim has been made that they are less virulent forms. 
While this may be true, the detection of Much's granules is not gen- 
erally accepted as a reliable clinical method because of the difficulty 
of differentiating them from other organisms or granular material . It 
is recognized that cheesy material contains a wealth of chromatin 
particles emerging from the leukocytes in all stages of degeneration. 
While some of Much's granules may be degenerated tubercle bacilli, 
others doubtless have their origin in degenerated leukocytes. 

Antiformin Method of Preparing Sputum for Examination.— When 
tubercle bacilli are present in small numbers, they may be difficult to 
demonstrate with the foregoing methods. Numerous ways have been 
suggested for the digestion and concentration of the sputum. A 
reagent is required which will dissolve the cells, mucus, and protein 
material and many of the bacteria, but will not affect the tubercle 
bacillus. The resulting digested fluid is centrifugalized so as to con- 
centrate the tubercle bacilli in the sediment. Of the various methods, 
the one employing antiformin is preferable. 

Procedure. — Place equal quantities of sputum and antiformin in a 
small bottle provided with a tight-fitting rubber stopper. All utensils 
should be thoroughly clean, and only freshly distilled water employed 



358 THE EXAMINATION OF SPUTUM 

on account of the possibility of introducing acid-fast bacilli in old 

distilled water or tap water. Shake the mixture thoroughly until of 
tliin consistency and then pour it into a centrifuge tube. After 
centrifugalization decant the supernatant fluid and fill the tube with 
sterile distilled water in order to wash the sediment free of alkali. 
Again centrifugalize, pour off the salt solution, spread the sediment on 
a slide, dry it, fix with heat, and stain in the usual way for tubercle 
bacilli. It may be necessary to add a drop of Mayer's egg-albumen to 
the sediment to make it adhere to the slide. 

The writer's personal experience has been that this method affords 
no better results than a careful search for caseous particles according 
to Sir Andrew Clark's method. 

Cultivation of B. Tuberculosis (Petroff's Method).— In attempting 
to cultivate the tubercle bacillus, PetrofT takes advantage of the fact 
that treatment with 3 per cent, sodium hydrate destroys staphylococci 
and streptococci, and that gentian violet in the culture medium inhibits 
the growth of many contaminating organisms, especially the moulds 
and the hay bacillus. 

The sputum should be obtained in as nearly an aseptic manner as is 
possible, directing the patient to use an antiseptic mouth wash before 
expectorating. Mix equal parts of sputum and 3 per cent, sodium 
hydroxide, shaking well. Incubate the mixture at 38° C. for from fifteen 
to thirty minutes, until it has become of thin consistency. Then add 
hydrochloric acid until the mixture is neutral to litmus and centri- 
fugalize at high speed for ten minutes. Pour off the supernatant fluid 
and inoculate the sediment into tubes containing Petroff's medium. 
The tubes should be left in the incubator for a few days until the 
moisture has evaporated or has been absorbed by the medium. Then 
the cotton stoppers should be paraffined, or they should be removed 
and sterile paraffined corks inserted in their places. The temperature 
should be kept at 38.5° C. as constantly as is possible. 

Petroff's medium is prepared by mixing one part of meat juice, two 
parts of egg (both white and yolk), and gentian v T iolet in a quantity 
sufficient to make 1 part of 10,000 parts of media. The ingredients 
are prepared as follows : 

I. Meat Juice.— Infuse 500 gm. of ground beef or veal in 500 cc. of a 
15 per cent, aqueous dilution of glycerine. At the end of twenty-four 
hours squeeze out the meat in a sterile press, collecting the infusion in a 
sterile glass beaker. 

II. Eggs.— Allow the eggs to remain in 70 per cent, alcohol about 
fifteen minutes to sterilize the shells. Break the shells and pour into 
a sterile beaker. Mix well with a sterile glass rod, and filter through 
sterile gauze. 

III. Geritian Violet.— Prepared a 1 per cent, alcoholic solution and 
add to the medium to give a dilution of 1 : 10,000. 

When ingredients have been mixed in the proper proportions, the 
medium is tubed and inspissated, at 85° C. on the first day in order to 



MICROSCOPIC EXAMINATION 



359 



solidify it, and at 75° C. on the two following days, for periods of one 
hour. 

The length of time required for a visible growth to appear varies from 
five to fifteen days, the average being nine to ten days. The growth is 
seen as a granular, heaped-up, yellowish streak. A slide preparation 
should be made and stained by the customary technic as outlined above. 
Should subcultures be desired, it is unwise to make them on medium 
containing gentian violet, since the dye ultimately retards the growth. 

Staining the Influenza Bacillus.— The influenza bacillus stains very 
slowly with ordinary dyes. It is small and irregular in size, especially 
in length. It may appear as a very small diplococcus or short diplo- 
bacillus (Fig. 111). The longer forms stained with Gram's stain may 
resemble "safety-pins," the ends being a faint violet, the central por- 
tion taking the counter stain. The organisms occur most frequently 
in clumps. 




Fig. 111. — Influenza bacilli in sputum. X 1000. (Lord.) 

Slides should be made by the "impression method." Gram's stain 
is recommended. These may be counterstained for five minutes with 
diluted carbol-fuchsin (i. e., 1 part of carbol-fuchsin plus 9 parts of water 
or safranin) . 

Cultivation of Bacillus Influenzae.— A sterile container should be given 
to the patient for the collection of the sputum. Two small beakers 
should be partly filled with sterile normal sodium chloride solution. 
A dense clump of sputum should be secured with a sterile platinum 
wire and washed by moving it vigorously about in succession in each 
dish of sterile salt solution. When the washing has been completed, 
the mass of sputum is drawn in parallel streaks across the surface of 
fresh blood-agar plates or tubes, preferably the former. These plates 
or tubes should be prepared by adding, under aseptic precautions, a 
few drops of fresh blood to melted agar at 40° C, after which the agar 
should be allowed to solidify. 



360 THE EXAMINATION OF SPUTUM 

The influenza colonies, which are small and have a "dew-drop" 
appearance, appear in about forty-eight hours. Sub-plants may be 
made from them to fresh blood-agar tubes. If a growth appears, 
slides should be examined to ascertain whether or not the organisms 
have the characteristic morphology and staining appearance. 

Eckers Method.— The bronchial secretions are mixed with 0.5 per 
cent, solution of sodium taurocholate. After standing for at least 
twenty minutes, streaks are made on blood-agar plates. Minute, 
colorless, dew-drop colonies appear after twenty hours' incubation at 
37° C. Bile salts have no effect on influenza bacilli, while they dissolve 
pneumococci and inhibit the growth of other accessory organisms. 

Avery's Method.— While the influenza bacillus may be recovered by 
this method, overgrowth with other organisms is more than likely so 
that isolation is often difficult. The following method is to be pre- 
ferred. Avery has called attention to the fact that isolation of the 
influenza bacillus from nasal or throat cultures is unsatisfactory, since 
the organism is usually found in association with other bacteria which 
grow much more luxuriantly. He suggests the addition to the medium 
of sodium oleate since this prevents the growth of certain Gram- 
positive organisms, notably the pneumococcus and streptococcus, 
while the growth of the influenza bacillus is enhanced. 

The medium may be prepared as follows: Two yer cent, meat 
infusion agar is made up in the usual way. The reaction should be 
from 0.3 to 0.5 acid to phenolphthalein. The initial hydrogen-ion 
concentration of the agar should be from 7.3 to 7.5 pH. A two per 
cent, solution of neutral sodium oleate is made in distilled water, sterilized 
in the autoclave, and kept on hand as a stock solution. Of this solution 
5 cc. are added to 95 cc. of the agar, giving a concentration of .1 : 1000. 
Sterile human blood or rabbit's blood is used. The blood is defibrinated 
and centrifugalized. The serum is withdrawn with a sterile pipette 
and the cell residue is made up to the original volume with nutrient 
broth. The blood suspension should be added while the agar is at 
about 40° C. The final formula calls for the following: 

2 per cent, meat infusion agar 94 cc. 

2 per cent, solution of sodium oleate 5 " 

Suspension of red blood cells 1 " 

If nutrient broth be desired, oleate hemoglobin bouillon may be pre- 
pared by the same formula by substituting broth for agar. 

Cultures should be taken from the nasopharynx by the West tube 
or from the throat by direct swabbing. They should be streaked on 
the surface of the medium according to the method outlined elsewhere. 
(See page 411.) Sputum, obtained in a sterile bottle as free from 
mouth contamination as possible, may be streaked across a plate with 
a sterile cotton swab. 

Diplococcus Pneumoniae.— The pneumococcus may be identified in a 
preliminary way by morphology and staining characteristics. Cover- 



MICROSCOPIC EXAMINATION 



361 



slips are prepared from the sputum. One should be stained with 
Gram's stain and one with Hiss' or Welch's capsule stain. The sero- 
logical type may be determined as outlined in subsequent paragraphs. 
The organism is a fairly large, lance-shaped coccus, occurring in 
pairs (Fig. 112). It is non-motile and possesses no flagellar. The 
shape may vary from an elongated, almost bacillary form, to a dis- 
tinctly spherical one. Short or long chains may be seen. The organ- 
isms are Gram-positive and are surrounded by a capsule which usually 
includes a pair of cocci. The capsule may be absent, however, its 
presence depending somewhat upon the environment of the organism. 
The most suitable conditions for capsule development are found when 
growth has taken place in the body fluids of man or animal. Capsules 




Fig. 112. — Pneumococci in fresh sputum. (Lord.) 

are usually demonstrable around the organisms found in the sputum, 
and are not found when growth has taken place in artificial media, 
except milk or those media containing serum. Even under unfavor- 
able conditions of cultivation, however, capsules may be demonstrated 
by using serum (such as beef or rabbit-serum) as a diluent when spread- 
ing organisms upon the cover-slip preparatory to staining. 

The pneumococcus does not grow readily upon ordinary media. The 
media should be neutral or slightly acid in reaction. Meat-extract 
media are not as well suited as are media made from a basis of an 
infusion of fresh meat. Growth may be aided by the addition to the 
medium of defibrinated blood, or animal or human serum, or by the 
addition of certain substances such as dextrose and glycerine. 



362 THE EXAMINATIOX OF SPUTUM 

To differentiate the pneumococcus from the streptococcus, the 
presence or absence of capsules may be helpful. This criterion is not 
final since pneumococci may show no capsules under unfavorable 
conditions. . As a means of differentiation, two reactions may be used. 
When Hiss' inulin-serum-water medium is used as a medium, pneumo- 
cocci break down the inulin causing acid formation and the reddening 
of the litmus, while streptococci do not. Sterile bile dissolves most 
pneumococci but not streptococci. 

The diplococcus of pneumonia is found occasionally in the mouths 
of healthy people, in the sputum of acute and chronic bronchitis, at 
times in the sputum from cases of pulmonary tuberculosis, and in the 
sputum of pneumonic conditions, where it is a causative factor. 

Determination of Type of Pneumococcus.— The determination of type 
is of great importance since treatment with serum is advisable at 
present only in infections due to a Type I organism. The facilities 
needed are simple, including those necessary for bacterial cultivation 
(media, incubator, etc.), for collection and microscopic examination of 
sputum, and for mouse inoculation and autopsy. The technic of 
typing has been developed and described very fully by Avery, Chicker- 
ing, Cole, and Dochez. Their method is presented here. For typing, 
the organisms may be recovered from the sputum, spinal fluid, 
empyema fluid, and by blood culture or lung puncture. 

Culture Media for the Pneumococcus.— For the cultivation of the 
pneumococcus, several requirements should be met: (1) The media 
should be prepared from infusions of fresh meat and not from beef 
extracts. (2) The reaction should be from 0.3 to 0.5 acid to phenol- 
phthalein. (3) Excessive heat must be avoided in sterilizing the 
media. It is preferable to sterilize by the Arnold method on three 
successive days for twenty minutes each time. 

The following directions are given by Avery, Chickering, Cole, and 
Dochez for the preparation of media : 

" One pound of lean chopped beef is allowed to infuse in, a liter of tap 
water over night on ice. The unfiltered infusion is boiled for thirty 
minutes, filtered through paper, and the loss by evaporation is made up 
by the addition of water. One per cent, peptone and 0.5 per cent, 
sodium chloride are now added. 1.5 per cent, agar may be added at 
this point if agar is desired. The mixture is allowed to boil for two 
minutes in the case of broth, or, if for agar, until the agar is dissolved. 
Titration to the neutral point with normal sodium hydrate is then 
carefully carried out. Media are boiled for six or seven minutes, made 
up to volume, and filtered clear, and sterilized in the Arnold sterilizer 
for twenty minutes on three successive days. The media should finally 
titer 0.3 to 0.5 to phenolphthalein." Bob-veal may be used. Medium 
may be filtered through glass wool instead of filter paper. 

Blood Agar. Media may be enriched by the addition of small 
amounts of defibrinated blood, obtained from a rabbit by puncture of 
an ear vein or by direct heart puncture. The blood should be drawn 



MICROSCOPIC EXAMINATION 363 

directly into a sterile container containing small glass beads and 
defibrinated by shaking. The blood may be kept for a week or more 
in a refrigerator. Aseptic technic should be employed. Three or four 
drops of this defibrinated blood are added to 4 or 5 cc. of bouillon or 
agar. 

Sterilization of Bile for Testing the Solubility of Pneumococci.— Tresh. 
ox-bile is obtained from the slaughter house and autoclaved for 
twenty minutes at 15 pounds pressure. The precipitate which occa- 
sionally is seen after heating may be removed by filtering. The 
filtered bile should be autoclaved again. When bile is not available, 
a sterile 10 per cent, solution of sodium taurocholate may be used. 
Solubility is determined by adding to a plain broth culture one-fifth 
its volume of ox-bile or sodium taurocholate solution. 

Procedure for Typing. — Collection of Specimen.— Sputum from the 
deeper air passages should be expectorated into a sterile, wide-mouthed 
container, as free from saliva as possible. It should be sent to the 
laboratory and examined with as little delay as possible. When delay 
is unavoidable, the sputum should be kept on ice. 

Preliminary Examination.— Make films and stain them with Gram's 
stain, with Hiss' capsule stain, and for tubercle bacilli. Avery and his 
co-workers direct attention to the fact that it is often possible to 
identify Type III organisms by staining methods, since they possess 
especially large, distinct capsules. 

Mouse Inoculation.— Select a portion of the sputum about the size 
of a kidney bean and wash it through three or four changes of sterile 
physiological salt solution in sterile Petri dishes. If the specimen be 
exceedingly friable or if it be almost free from contaminating organisms, 
washing may be omitted. The particle of sputum, washed or unwashed 
as the case may be, is placed in a small sterile mortar and ground up 
with 1 cc. of the sterile physiological salt solution to form a homo- 
geneous emulsion which will pass through a hypodermic needle. 

One-half to 1 cc. of the emulsion is injected by means of a sterile 
syringe into the peritoneum of a white mouse (Fig. 113). The pneumo- 
coccus grows rapidly, while practically all other organisms (with the 
exception of Friedlander's bacillus, the influenza bacillus, and occa- 
sionally others) die. The blood stream is usually invaded early by 
pneumococci and not by the other organisms. The time required for 
a sufficient growth in the peritoneum varies from five to twenty-four 
hours, averaging six to eight hours with Types I, II and III. When the 
mouse appears ill, a little of the peritoneal exudate is secured by 
puncturing the peritoneum with a sterile capillary pipette, after 
sterilizing the skin and making a small incision through it with a sterile 
knife. The exudate is removed with the pipette and spread upon a 
slide. It should be stained with Gram's stain and examined to deter- 
mine if there be a sufficient growth of pneumococci. If the growth be 
only moderate or if there be many other organisms present, time should 
be allowed for more abundant growth. 



364 



THE EXAMINATION OF SPUTUM 



Autopsy of the Mouse.— This should he performed as soon as the 
mouse dies or is killed. The peritoneal cavity is opened with suitable 
aseptic precaution and cultures are made from the peritoneal exudate 




Fig. 113. — Intraperitoneal inoculation of mouse. Rockefeller Institute Monograph No. 7. 




Fig. 114. — Method of collecting peritoneal washings from sputum-inoculated mouse. 
Rockefeller Institute Monograph No. 7. 



MICROSCOPIC EXAMINATION 365 

in plain or 1 per cent, dextrose broth and on one-half of a blood agar 
plate. Films are made from the exudate and are stained as with 
Gram's stain and Hiss' capsule stain. Then 4 or 5 cc. of sterile physio- 
logical saline solution are placed in a test-tube. By means of a sterile 
1 cc. pipette fitted by a rubber nipple, a small portion of the solution 
is used for washing out the peritoneum (Fig. 114). The purulent 
material so obtained is mixed with the bulk of salt solution in the test- 
tube and the process repeated several times, when the washings are 
transferred to a sterile centrifuge tube. Finally the heart is opened 
and cultures are made in plain broth and on the other half of the blood 
agar plates. 

Rapid Cultural Method.— On account of the difficulty in securing 
white mice, Avery devised a rapid cultural method. A selected puru- 
lent kernel of sputum should be washed and emulsified with broth as 
described in a foregoing section. The emulsion is inoculated into a 
centrifuge tube containing about 4 cc. of glucose-blood broth. 1 

After inoculation of the glucose-blood-broth, the tube is placed in a 
water-bath at 37° C. for five hours when a loopful of the culture is 
streaked on a blood agar plate in order to isolate the pneumococcus in 
pure culture for subsequent confirmatory determination of type by the 
agglutination method. The red blood cells are removed by centri- 
fugalizing at low speed for three or five minutes. Erythrocytes should 
settle to the bottom of the tube and bacteria remain in the supernatant 
fluid. Three or four cc. of supernatant bacterial suspension are 
transferred with a sterile pipette to a second sterile centrifuge tube 
containing about 1 cc. of sterile ox-bile. This is allowed to stand in a 
water- bath at 37° C. for about twenty minutes, or until the pneumo- 
cocci have been dissolved. This bile solution of pneumococci is used 
in the precipitin tests. When bile is not available, the pneumococcus 
suspension may be used for macroscopic agglutination tests. 

Avery found that the results obtained with his method checked very 
closely with those obtained with the mouse inoculation method, especi- 
ally when due pains were taken to select suitable portions of sputum, 
to wash them thoroughly, and to free the pathogenic organisms in the 
center of the sputum kernels by thorough grinding in the mortar. He 
thinks, however, that while the results are obtained rapidly, they should 
be checked by the mouse-inoculation method when mice are available. 

Determination of Type of Isolated Organism by the Agglutination Method. 
—If the organisms be obtained in pure culture, the agglutination 
method should be used. The tube containing the peritoneal wash- 
ing is centrifugalized at low speed for a few minutes to throw down 
cells and fibrin. The supernatant bacterial suspension is pipetted off 
into another sterile centrifuge tube, which is centrifugalized at high 

1 Broth is made as outlined in the foregoing paragraphs and to each 100 cc. are 
added 5 cc. of sterile defibrinated rabbit's blood and 5 cc. of a sterile 1 per cent, glu- 
cose solution. A glucose solution should have been sterilized in the Arnold sterilizer 
by the fractional method at 100° C. on three successive days. 



366 THE EXAMINATION OF SPUTUM 

speed to throw down all the organisms. The supernatant fluid, which 
should be almost clear, is pipetted off and discarded. To the sediment 
is added sterile physiological saline solution in a sufficient quantity to 
make a moderately heavy suspension which will have the density of a 
good eighteen-hour broth culture of pneumococcus. The suspension 
is used for the agglutination tests, placing 0.5 cc. in each of the small 
test-tubes. To avoid the confusion of cross-agglutination, proper dilu- 
tion of sera is important. This varies somewhat with different lots of 
serum. Five test-tubes are set up as follows: 

Tube 1 0.5 cc. Serum I (1: 20 dilution) + 0.5 cc. bacterial suspension. 

Tube 2 0.5 cc. Serum II (undiluted) + 0.5 " " " 

Tube 3 0.5 cc. Serum II (1: 20 dilution) + 0.5 " 

Tube 4 0.5 cc. Serum III (1: 5 dilution) +0.5 " 

Tube 5 0.1 cc. Sterile ox-bile + 0.4 " " " 

The tubes are incubated in a water-bath for one hour at 37° C. 
When agglutination occurs, there is a rapid clumping of the organisms, 
which may be observed when the tubes are shaken gently. If there 
be no agglutination in any of the tubes and if the organisms in tube 5 
dissolve with bile, the pneumococcus is classified as a Type IV. 

Determination of Type by the Precipitin Method.— When pneumococci 
are present in the washings from the peritoneum together with a large 
number of contaminating organisms, agglutination tests are unsatis- 
factory and the precipitin method should be given preference. The 
soluble substance liberated by the pneumococci during its growth gives 
a specific precipitin reaction with homologous anti-pneumococcus 
serum. The peritoneal washings obtained as described in the para- 
graph on the "Autopsy of the Mouse," are placed in a centrifuge tube 
and the tube is run in the centrifuge at high speed to throw down the 
cells, bacteria, and fibrin. The clear supernatant fluid is pipetted off 
and used for the precipitin tests. One-half cc. is placed in each of the 
test-tubes which are set up as follows : 

Tube 1 0.5 cc. Serum I (1: 10 dilution) + 0.5 cc. supernatant peritoneal washings. 

Tube 2 0.5 cc. Serum II (undiluted) + 0.5 " 

Tube 3 0.5 cc. Serum II (1: 10 dilution) +0.5 " " " " 

Tube 4 0.5 cc. Serum III (1: 5 dilution) +0.5 " 

Incubation is usually unnecessary since the specific precipitin reac- 
tion occurs at once in the tube containing homologous serum. The 
contact ring should be noted. The reason for employing two dilutions 
of Serum of Type II is to distinguish between the Type II pneumococcus 
and its sub-groups. The sub-group pneumococci give a reaction only 
with the undiluted serum while the pneumococci of Type II give a 
precipitation in both tubes. When a negative reaction occurs in all 
tubes, the organism is classed in Group IV. 

Confirmation of Findings. — The results obtained with the peritoneal 
washings should be regarded as only preliminary and should be con- 
firmed by carrying out similar tests with the pure culture obtained 



MICROSCOPIC EXAMINATION 



367 



from the heart's blood. The technic is the same as that outlined for 
peritoneal washings. 

Determination of Type III Pneumococcus.— Pneumococcus mucosus 
(or Type III) can often be identified on morphological and cultural 
grounds. It is larger, not so definitely lanceolate, and broader than 
the other types (Fig. 115). The capsule is large and stains well with 
the capsule stains, often showing with the counterstain of Grain's 
stain. Cultures on blood-agar are moist, mucoid, and confluent, and 
the peritoneal exudate of the infected mouse is also sticky and mucoid. 







?■'" 


■JK^> 



Fig. 115. — Streptococcus mucosus capsulatus in sputum. (Lord.) 



Micrococcus Catarrhalis.— This organism is found occasionally in 
the sputum of patients suffering with catarrhal affections of the upper 
respiratory tract. It is only slightly pathogenic. On morphological 
grounds it cannot be distinguished from the gonococcus or meningo- 
coccus, for it is a small, biscuit-shaped, Gram-negative diplococcus 
(Fig. 116). Its ready growth upon ordinary media, however, furnishes 
a point of differentiation from the gonococcus, which grows only upon 
special media. Differentiation from the meningococcus is not so easy, 
since this coccus occasionally grows readily upon ordinary media. 
The growth of the micrococcus catarrhalis upon ordinary culture 
media is heavier and the colonies are granular and distinctly white, 
while those of the meningococcus are translucent, finely granular, and 
grayish-white. Agglutination reactions are helpful in making a 
differentiation. The differentiation assumes great significance in the 
examination of cultures from the nose and throat. 

Streptococcus and Staphylococcus.— Streptococci and staphylococci 
of all varieties may be found in the sputum, not only in cases of acute 



368 



THE EXAMINATION OF SPUTUM 



and chronic bronchitis, but as secondary invaders in advanced tubercu- 
losis. Streptococci may be found in acute lobular and bronchopneu- 




Fig. 116. — Micrococcus catarrhalis in smear from sputum. (Lord.) 




Fio. 117. Friedlander's bacillus in sputum. (Lord.) 



monia, where they may be the causative factor. When one desires 
to cultivate the streptococci and to study the cultural characteristics, 



THE SPUTUM IN DISEASE 369 

the sputum should be washed and ground to an emulsion as directed 
in the foregoing paragraphs which describes the typing of pneumococci. 
Cultures may then be made in ordinary media and on blood agar. 
(See Bacteriology of Blood.) 

Bacillus Mucosus Capsulatus.— Friedlander's bacillus is present in a 
comparatively small number of the cases of lobar pneumonia. The 
infection is extremely severe and usually fatal. The organism is a 
short, plump bacillus, some of the shorter and thicker forms resembling 
cocci (Fig. 117). It is non-motile, forms no spores, stains readily with 
ordinary stains, and Gram-negative. The thick capsule is clearly 
marked and is readily demonstrated when the organism has been 
obtained from body exudates, often when grown upon agar. The 
bacillus is readily cultivated upon ordinary media. 

Bacillus Smegma.— The smegma bacillus is of importance from the 
standpoint of differentiating it from the tubercle bacillus. Its occur- 
rence has been reported in the sputum and in the material squeezed 
out of the tonsillar crypts. While acid-fast, the bacilli do not retain 
the stain in the presence of acid as tenaciously as tubercle bacilli. It 
is generally thought that Pappenheim's rosolic acid method of decolor- 
izing is adequate for differentiation. 

THE SPUTUM IN DISEASE. 

Acute Bronchitis. — In the earlier stage of acute bronchitis, the sputum 
is white, contains a few leukocytes and red blood cells, epithelial cells, 
and mucus, and often a number of varieties of organisms. Later, the 
color changes to a yellow or even a greenish tint and microscopic 
examination shows many leukocytes and a few red blood cells. Smears 
made directly from the sputum may show many types of organisms, 
but often only one or two types of microorganisms are found after the 
purulent mass is washed and ground to obtain only the organisms from 
the lower portions of the respiratory tract. The possible offending 
organisms include the pneumococcus, streptococcus, micrococcus 
catarrhalis, the influenza bacillus, and occasionally Friedlander's 
bacillus or even the colon bacillus. In making a diagnosis of influenza, 
it is advisable to culture the organism from the sputum. 1 

Chronic Bronchitis. —The sputum is extremely variable in both 
amount and appearance. In some cases, there is none. Frequently 
it is abundant, mucopurulent or purulent in appearance, yellow or 
green in color, and contains many degenerated leukocytes and alveolar 
epithelial cells. Practically any of the organisms mentioned as possible 
invaders in bronchitis may be found. 

1 The role which the influenza bacillus played in the great epidemic of the latter part 
of 1918 is a moot question. Lord, Scott and Nye found 78 per cent, positive influ- 
enza cultures in epidemic cases. Pritchett and Stillman using Avery's oleate blood 
agar medium, cultivated the B. influenzae in 93 per cent, of the cases of influenza and 
bronchopneumonia. In interpreting these lesults, it is to be borne in mind that 
the influenza bacillus is a common invader of the respiratory tract, that it is found in 
normal individuals, and that it often occurs even in cases of pulmonary tuberculosis 
with the tubercle bacillus. 
24 



370 THE EXAMINATION OF SPUTUM 

Bronchial Asthma.— The sputum is usually thick and tenacious and 
often is expectorated in the form of little balls or gelatinous masses, 
the "perles" of Laennec. Curschmann's spirals, eosinophilic cells, and 
( harcot-Leyden crystals are often seen. 

Acute Lobar Pneumonia. — In the early stages, the sputum may be 
mucoid in character but the appearance soon changes and it becomes 
blood-tinged, "rusty," tenacious or sticky. It is frequently very 
viscid. Occasionally the sputum may be fluid and brown in color, 
when it is spoken of as "prune-juice sputum." During the stage of 
resolution it becomes yellow, thinner and more abundant, and the 
cells are more numerous. At times it is green. The pneumococcus 
may be seen in cover-slip preparations when suitably stained and may 
be cultivated by methods which have been described. 

Gangrene of the Lung.— The sputum has a very fetid, offensive 
characteristic odor. It is usually fluid and separates into three layers, 
of which the lower is a heavy sediment; the middle, fluid and green or 
brown in tint; and the upper, frothy and thick. At times portions of 
lung tissues are expectorated. Microscopically, fatty acid crystals, 
bacteria, a few cells, granular material, and elastic tissue are found. 

Fibrinous Bronchitis. — In chronic idiopathic bronchitis, casts of a 
certain portion of the bronchial tree may be expectorated. At times 
the casts are rolled up and mixed with blood and mucus, or they may 
be seen as large branching structures. They consist mainly of fibrin, 
and contain leukocytes and epithelial cells. Fibrinous bronchitis is 
seen occasionally in the course of some of the acute infections, notably 
diphtheria, typhoid fever, pneumonia and the eruptive fevers. 

Chronic Passive Congestion.— The sputum is often tenacious and blood- 
tinged. On microscopic examination, "heart failure cells" and red 
blood cells may be demonstrated. 

Acute Pulmonary Edema.— The sputum is copious, frothy, and is. 
often tinged a bright pinkish color from the admixture of blood. 

Malignant Disease of the Lung.— There is nothing especially charac- 
teristic about the sputum though occasionally bits of the new growth 
are expectorated. When found, the}' should be sectioned and examined 
histologically. 

Pulmonary Tuberculosis.— The sputum varies greatly in quantity, 
appearance, and in the number of tubercle bacilli which it contains. 
In acute miliary tuberculosis it may be slight in amount, similar in 
appearance to that seen in acute bronchitis, and devoid of tubercle 
bacilli. In early cases of pulmonary tuberculosis, the quantity is often 
small, and expectoration may be limited to the early morning. Blood 
streaks are seen at tinies; indeed, a profuse hemorrhage may be the first 
symptom. As the lung tissue breaks down sputum is mucopurulent, 
more copious in amount and contains clastic tissue and broken down 
lung tissue. Advanced cases in which cavity formation has taken 
place show so-called "nummular sputum," spherical masses of pus 
floating in thin fluid. Such sputum often has an offensive odor, and 
the purulent portions may contain large numbers of tubercle bacilli. 



CHAPTER VI. 

EXAMINATION OF BODY FLUIDS, EXUDATES AND 
MISCELLANEOUS METHODS. 

EXAMINATION OF SPINAL FLUID. 

Technic of Spinal Puncture.— The puncture must be carried out under 
aseptic conditions. Especial attention should be given to the pre- 
liminary arrangement of the patient, who lies on his right side on the 
extreme edge of the bed, with the lower portion of the back as nearly 
straight in both planes as possible, and the knees drawn up so that the 
thighs are at right angles to the trunk. Both shoulders should be at 
right angles to the surface of the bed, in order to prevent a sagging of 
the body and consequent curving of the spine in the lumbar region. 




Fig. 118. — Showing point for lumbar puncture in median line in the first interspace 
below the level of the iliac crests. It is impossible to show the correct position of the 
needle, which should be inserted at a right angle in both planes. 



Often a small pillow placed in the curve between the crest of the ilium 
and the costal margin is useful in keeping the spine straight. The skin 
of the entire lumbar area is carefully cleansed by scrubbing with 
tincture of green soap, ether, alcohol, and finally by applying tincture 
of iodine. Anesthesia is produced by infiltrating the skin and sub- 
cutaneous tissues with j^- solution of procain by means of a sterile all- 
glass syringe. The procain solution may be sterilized by boiling. For 
the puncture, a needle fitted with an obturator is desirable. 



372 



EXAMINATION OF BODY FLUIDS AXD EX (DATES 



The intervertebral space at the level of the iliac is the one usually 

selected for puncture, though sometimes the space immediately above 
is somewhat easier to enter (Fig. 118). The lumbar puncture needle 
is inserted in the mid line toward the lower border of the intervertebral 
space, keeping it at right angles to the body in both planes, when it should 
pass into the canal without touching bone in its course. If the space 
below the line of the iliac crests be selected, the point of the needle 
should be directed slightly toward the head. When it is felt that this 
space has been entered, the obturator should be withdrawn. Should 
no fluid appear, the obturator is replaced and the needle inserted a little 
farther. Failure to obtain fluid is often due to the fact that the point 
of the needle has been directed downward or to the right of the canal 
so that the needle may be partially withdrawn and the butt end 
depressed in order to raise the point of the needle, which on reinsertion 



ii \£- ^> i ^- V 

1 I I INN 



1 2 3 * £ 6 7 8 9 10 

Fig. 119. — Colloidal gold reaction curves. Broken line represents paretic curve; 
solid line, curve in luetic zone. Tube numbers are represented on abscissa line and 
depth of color change on ordinate line. 



usually reaches the desired destination. Unless pressure be greatly 
increased or serum be injected, not more than 7 or 8 cc. should be 
removed. (For an excellent discussion of methods of performing 
lumbar puncture, see article by Joseph C. Regan.) 

Precautions. — When brain tumor is suspected, much care should be 
exercised not to decompress too rapidly and to take only a small 
amount of fluid. Under all circumstances, the patient should be kept 
flat on the back or side in bed for at least twenty-four hours, not raising 
the head from the pillow for any purpose. 

With all possible precautions, a severe headache, the so-called 
"puncture headache," appears occasionally. This may last for days, 
even as long as three weeks. Apparently there is no effectual method 
of treatment. The patient is usually perfectly comfortable when in 
the recumbent posture but as soon as the head is raised complains of a 



EXAMINATION OF SPIXAL FLUID 373 

throbbing sensation at the base of the skull and back of the neck and 
at times of nausea and vomiting. 1 

If a determination of pressure be desired, a long piece of glass tubing 
with internal diameter of 1 mm. may be attached to the needle by a 
piece of rubber tubing. The spinal fluid is allowed to ascend as far as 
it will go. The height to which it ascends may be marked on the tube 
by an assistant with adhesive tape or a wax pencil and the distance 
measured in millimeters. The determination t of pressure is hardly 
worth while from a practical point of view. When it is greatly in- 
creased, as in tuberculous meningitis, the fact is quite apparent, since 
the fluid spurts out in a stream, often for a distance of several feet. 
Normally it comes out drop by drop, though the first cubic centimeter 
or so may come in an almost continuous stream. The manometer 
readings are not absolutely accurate since the level of the fluid depends 
greatly upon the relative position of the patient's head. 

Routine Examination of the Spinal Fluid.— The procedure varies some- 
what with the clinical findings. When syphilis of the central nervous 
system is suspected, the following would be an appropriate order for 
the tests : 

1. Gross appearance (color, turbidity, presence of blood, etc.). 

2. Cytological examination. 

3. Chemical examination (globulin and Lange tests). 

4. W^assermann reaction. 

When an acute infective meningitis is suspected, the following should 
be the order of the examination : 

1. Observation of gross appearance (color, turbidity, presence of 
macroscopic pus, etc.). 

2. Inoculation of media. 

3. Removal with a sterile pipette of about 1.5 cc. for cell count. 

4. Centrifugalization of a portion for a cytological and bacterio- 
logical study of cells. 

5. Incubation of remainder of uncontaminated fluid in original 
container at 37° C. 

Gross Appearance.— Ccjhr.— Normal spinal fluid is colorless, and 
in the gross cannot be distinguished from distilled water. Its specific 
gravity is given as 1.006 to 1.007. Xanthoeromia (canary-yellow 
tint) may be seen after intracranial hemorrhage. It is usually due to 
changed blood, and is generally accompanied by an increased amount 
of globulin. Nonne's syndrome (yellow color, no increase in the cell 
count, and an excess of protein), may be produced by localized oblit- 

1 A plausible theory has been advanced by MacRobert to account for these head- 
aches. The dura is a rigid, tough membrane without elastic tissue. When it has been 
punctured, the hole persists, sharp and clean-edged, unless closed by soft arachnoid 
tissue which may fill it and so block it. This closure may not occur since the sub- 
arachnoid tissue may be dragged through the hole by the departing needle and so form 
a spout or wick for the drainage of the cerebrospinal fluid. This constant drainage 
permits a marked diminution in the amount of fluid, and the base of the brain is 
deprived of its supporting cushion of fluid. 



374 EXAMINATION OF BODY FLUIDS AND EXUDATES 

(■ration of the pia-arachnoid space, forming a lower cul-de-sac with no 
communication with the space above. 

Turbidity. — When turbidity is present, it is due to a high cellular or 

bacterial content. In most cases of cerebrospinal syphilis, the fluid is 
clear, but distinct turbidity may be seen with the high cellular content 
which sometimes accompanies active syphilitic meningitis. Fluids 
of this type may present the opacity of ground glass. 

Blood.— Blood may be seen when the tissues have been injured 
by the needle; in cases of cerebral hemorrhage when the area of hemor- 
rhage has communicated with the subarachnoid space ; and after frac- 
ture of the skull. Fluid drawn a considerable time after hemorrhage 
may show a canary-yellow color. 

Cytology.— The cells may be enumerated by the following method 
(Swift and Ellis) : The hemocytometer commonly used for enumer- 
ating white blood cells and the ordinary hemocytometer chamber are 
employed. Staining fluid (methyl violet, 5 B., 0.1 gm.; glacial acetic 
acid, 10 cc. ; distilled water, q. s. to 100 cc.) is drawn to 1 .0 mark and then 
aspirated into the pipette to coat the walls of the bulb. The pipette 
is filled with freshly drawn spinal fluid to the 11 mark, the mixture is 
thoroughly shaken and set aside for about fifteen minutes. At the 
end of this time the lymphocytes will have been stained sufficiently. 
Ten fields, each 10 mm. square and 0.1 mm. deep, are counted, follow- 
ing the routine described by the enumeration of leukocytes (see page 
38). The total, multiplied by --$- to correct for the dilution, gives the 
number of cells per cm. 

The count will be reduced considerably unless it be made very soon 
after the withdrawal of the fluid, since the lymphocytes disintegrate 
readily. The normal count varies from 1 to 6. An increased count is 
referred to as jAeocytosis . 

Differential Count.— The fluid is centrifugalized for fifteen or twenty 
minutes when the supernatant fluid is withdrawn with a capillary 
pipette. The residual drop is placed upon a slide, dried, fixed and 
stained with Wright's stain. The essential points in the technic are: 
slow drying of the sediment in the air with little or no heat; fixation 
with absolute methyl alcohol for two minutes. The application of the 
flame should be avoided to prevent rapid drying of the cells of lympho- 
cytic type. Normally, only lymphocytes are seen. 

( 'iiemk'al Examination.— Reduction of Fehling's Solution.— Normal 
spinal fluid contains a substance, presumably a sugar, which reduces 
Fehling's solution. It is absent in meningitis, but may be present in 
slight inflammatory conditions. Absence of reduction is of uncertain 
import. The test has little practical value. 

Globulin.— Globulin is found in normal spinal fluid, but only in 
traces, increased amounts accompanying practically all inflammatory 
conditions. 

Noguchi Butyric Acid Test.— Mix 0.2 cc. of spinal fluid (free of blood) 
in a test-tube with 1.0 cc. of 10 per cent, dilution of butyric acid in 



EXAMINATION OF SPINAL FLUID 375 

normal saline solution. Heat this to boiling and add 0.2 cc. of normal 
sodium hydrate. Heat the mixture again to boiling, and cool. Nor- 
mally a slight opalescence is obtained. A distinctly granular or rloccu- 
lent sediment indicates an increase in globulin. The importance of 
using absolutely pure butyric acid has been emphasized by Ross and 
Jones, who secured a large number of faulty negative results with the 
Noguchi test because of the use of impure acid. 

An arbitrary standard for reading the Noguchi reaction has been 
suggested by Swift and Ellis: 

Negative: opalescent to very faint haze. 

Plus-minus: faint haze to haze. 

One plus : fine granular precipitate. 

Two plus: heavy granular or coarse flocculent precipitate. 

Three plus : very heavy flocculent precipitate. 

Pandy's Test. — To about 1 cc. of a saturated aqueous solution of 
phenol is added one drop of spinal fluid. In the presence of an 
increased quantity of globulin, a bluish-white ring or cloud is formed 
immediately. 

Ross- J ones Modification of Nonne's Test.— In a test-tube are placed 
about 2 cc. of a saturated solution of ammonium sulphate. One cc. 
of spinal fluid is run into the test-tube from a pipette to form a layer 
over the ammonium sulphate solution. A white or gray ring marking 
the point of contact constitutes a positive reaction. 

Lange Colloidal-gold Reaction.— Normal spinal fluids will cause no 
change in properly prepared solutions of colloidal gold when suitably 
diluted with 0.4 per cent, solution of sodium chloride, which is of the 
proper concentration to keep the protein in solution. Abnormal fluids 
bring about partial or complete precipitation of the colloidal gold, 
producing color changes which may be graphically charted and are 
apparently specific for certain conditions. 

The following technic has been adapted from that published by 
Miller, Brush, Hammers, and Felton, with only minor changes. After 
a trial of many other methods, the writer has adopted this for his own 
use and has found it highly satisfactory. 

Solutions Required.— (1) Solution of colloidal gold, which must 
answer the following requirements : 

(a) It must be neutral to alizarin on the day it is used. 

(6) It must be absolutely transparent and of a brilliant orange-red 
or salmon-red color. 

(c) It must be "unprotected," that is, 5 cc. should be completely 
precipitated in one hour by 1.7 cc. of 1 per cent, sodium chloride 
solution. 

(d) It should give a typical paretic curve with a known paretic 
spinal fluid. 

(e) It should give no more than a No. 1 reaction in any tube with a 
known normal spinal fluid. 

(2) Sodium chloride solution, sterile, 0.4 per cent, prepared from 
C. P. NaCl and distilled three times. 



376 EXAMINATION OF BODY FLUIDS AND EXUDATES 

Preparation of Solutions.— The chemicals must be weighed with 
extreme accuracy. Lack of attention to detail will bring only failure. 

. [pparatus Required: 

Pyrex beakers, capacity 500 ce. 

Pyrex beakers, capacity 1000 cc. for making solution. 

Pyrex Florence flasks, capacity 2000 cc. for collecting water. 

Graduates, 100 cc. and 500 cc. 

Pipettes, two, 1 cc, graduated in r J- cc. 

Pipettes, four, 5 cc., graduated in 2 \ cc. 

Pipettes, four, 1 cc, one mark only. 

Pipettes, four, 10 cc graduated in ^ cc 

Thermometer of Jena glass. 

Bunsen burner, tripod, wire gauze with asbestos center. 

Copper still, tin-lined, with worm condenser of block-tin, including 
suitable supports and 4-tube Bunsen burner. 

Thick walled lipless test-tubes, about yi inch in diameter by 6 inches 
long. 

Rack with 12 holes of size to receive the test-tubes specified. 

Cleaning Glassware.— This is an essential step in the technic 
Beakers, flasks, pipettes, test-tubes, and graduates are treated first 
with a small amount of nitric acid, which is allowed to come in contact 
with the entire inner surface of the various vessels. This readily 
removes any precipitate. Tap- water is then used copiously for 
rinsing, which is continued with 2 or 3 changes of once-distilled water 
and finally rinsed with a little triple-distilled water just before use. 
The pipettes are drained by allowing them to stand upright on a piece 
of clean filter-paper. 

Reagents Required.— 1. Triple-distilled Water. 

The water which is used in making the solutions should be distilled 
three times. The same flask should be used throughout for receiving 
the distilled water. On each distillation, the first 100 cc. of distillate 
should be discarded. 

2. Gold Solution.— Gold chloride crystals should be obtained in 
small, hermetically-sealed glass ampoules. 

Gold chloride (Merck's yellow crystals) 1 gm. 

Water, triple-distilled, to 100 cc. 

This solution keeps for months when placed in a dark glass-stoppered 
bottle away from the light. 

3. Potassium Carbonate Solution.— This should be made up immedi- 
ately before use. 

Potassium carbonate (Merck's desiccated Hue label) ... 2 gm. 

Water, triple-distilled, to 100 cc. 

4. Formaldehyde Solution.— Merck's Blue Label (highest purity) 
40 per cent, solution formaldehyde should be used. A neutral reaction 
may be maintained by placing powdered marble-dust in the bottom 
of the bottle. The bottle is shaken occasionally and the powder 



EXAMINATION OF SPIXAL FLUID 377 

allowed to settle before use. The folowing solution should be made up 
just before use: 

Formaldehyde (40 per cent, solution) 1 cc. 

Water, triple-distilled, to 40 " 

5. Oxalic Acid Solution.— This solution also must be prepared just 
before it is needed for use in making up the colloidal-gold solution. 

Oxalic acid (Merck's blue label, crystals) 1 gm. 

Water, triple-distilled, to 100 cc. 

Preparation of the Colloidal-gold Solution.— In a beaker heat 1000 ce. 
of triple-distilled water, using a Bunsen flame, and protecting the 
beaker with asbestos-filled iron gauze. When the temperature reaches 
60° C, add 10 cc. of the gold chloride solution and 7 cc. of the potas- 
sium carbonate solution. At 80° C, stir the solution and add 10 drops 
of the oxalic acid solution. At 90° C, add the formaldehyde solution 
slowly, drop by drop, using only enough to produce an initial pink 
color. Usually about 5 cc. are required. The flame should be reduced 
to a point sufficient to keep the temperature at 90° C. until the color 
change appears, when it is removed. 

If satisfactory, the solution should correspond to the standards 
previously described. A yellow shimmer indicates an unsatisfactory 
solution. The solution should be transparent so that if a beaker con- 
taining 7.5 cm. of the solution be placed over a book-page, ordinary type 
may be read through the solution. 

When the correct amount of alkali has been employed, the solution 
usually remains colorless until it has reached a temperature of 90° C. 
When the solution, however, becomes definitely pink after the addition 
of oxalic acid, the formaldehyde should be added slowly and only 
until the color is intensified. If, on the other hand, no color appears 
after the temperature has been held at 90° C . for some time, formalde- 
hyde should be added drop by drop until a pink color is evident. When 
this color fails to appear, potassium carbonate solution may be added 
drop by drop until it is seen. 

Neutralization of the Solution.— When the solution answers all other 
requirements but is not neutral in reaction, it may be neutralized. 
The indicator to be employed is a 1 per cent, solution of alizarin red in 
50 per cent, alcohol. Added to a neutral colloidal gold solution, this 
gives a brownish-red tint, to an acid solution, a lemon-yellow color, 
and to one which is alkaline, a purple-red. Two drops of the indicator 
are placed in a test-tube with 5 cc. of the colloidal gold solution. If the 
color be brownish-red, the solution is satisfactory from the standpoint 
of reaction. If not, titration may be conducted in the following way. 
A rack is prepared with ten test-tubes, into each of which is placed 1 cc. 
of distilled water. Into the first test-tube, place 1 cc. -^j hydrochloric 
acid if the reaction of the solution be alkaline, or 1 cc. of -^ sodium 
hydrate if it be acid. Draw the contents of the first test-tube into a 
pipette and eject into the test-tube. Repeat this two or three times to 



378 EXAMINATION OF BODY FLUIDS AXD EXUDATES 

insure thorough mixing and then place 1 ee. in the second tube. Mix 
the contents of the second tube by drawing the fluid up into the pipette 
and ejecting it into the test-tube several times, and then place 1 ee. 
in the third tube. Repeat this procedure with each tube, omitting the 
tenth tube, and discarding 1 cc. of the mixture in the ninth tube. The 
amount of acid or alkali in each tube is readily calculated. In the first 
we have0.5 cc. ^ solution, in the second 0.25 cc, in the third 0.125 cc. 
and so on down. The tenth tube, containing- only colloidal gold solu- 
tion, serves for color comparison. To each tube add two drops of the 
indicator and 5 cc. of the colloidal gold solution to be tested. Select 
the first tube in which the neutral reaction (brownish-red tint) is 
obtained. This indicates the amount of acid or alkali which will have 
to be added to 5 cc. of solution to secure a neutral reaction. Divide 
this amount by 5 to find the amount necessary to neutralize 1 cc. 
and multiply it by the amount of solution to be neutralized. The 
corresponding amount of ^ acid or alkali should be added slowly while 
agitating thoroughly. Solutions should not be neutralized until they 
are at least forty-eight hours old. 

Performance of the Reaction .—When a .satisfactory solution is avail- 
able, the performance of the reaction itself is comparatively simple. 
Only uninfected, blood-free spinal fluids should be examined. 

Eleven test-tubes should be set up in a rack. In the first tube place 
1.8 cc. of 0.4 per cent. XaCl solution and 0.2 cc. of the spinal fluid to 
be tested. In each of the remaining tubes put 1 cc. of 0.4 per cent. 
NaCl. Mix the contents of the first tube well and transfer 1 cc. of the 
mixture to the second tube. Mix this, and with the same pipette take 
1 cc. and put in the third tube. Continue this with each tube up to 
and including the tenth. When the contents of this tube has been 
mixed, withdraw 1 cc. and discard it. The eleventh tube, containing 
only 0.4 per cent NaCl solution and no spinal fluid, serves as a control. 
Then add to each tube 5 cc. of the colloidal gold solution. Shake each 
tube separately and thoroughly. Set the rack aside, and take readings 
in thirty minutes and again in twenty-four hours. 

Readings.— In making the readings, each tube is given a number on 
a scale of 5 according to the extent of the color change. The following 
is the scale: 

Colorless 5 

Almost, colorless, hut a tinge of blue 4 

Blur 3 

Lilac or purple 2 

Reddish-blue 1 

Original salmon-red (unchanged) 

The readings are then recorded in the order in which the respective 
tubes stand: e. g., 5, 5, 5, 5, 4, 3, 2, 1, 0, 0, represents a curve in the 
"paretic zone," while within the "luetic zone" would be: 1, 1, 2, 3, 
2, 1, 1, 0, 0, 0, or 2, 2, 3, 4, 4, 3, 1, 0, in other words, where the greatest 
color change is reached in third, fourth or fifth tubes (Fig. 119). A 



EXAMINATION OF SPINAL FLUID 



379 



curve is referred to as falling within the " meningitic zone" when the 
depth of the color reaction is in the lower dilutions. 

Interpretation of the Findings. —The curve obtained in tabes and cere- 
brospinal lues fall in approximately the same zone. In treated cases 
the writer has sometimes found that the curve persisted in the luetic 
zone after the disappearance of the other laboratory findings. The 
paretic type of curve has been found in cases of multiple sclerosis where 
the Wassermann reaction was negative. The reaction is of distinct 
value in differentiating between paresis and syphilitic meningitis. 

Wassermann Reaction with Spinal Fluid.— The Wassermann reaction 
is of assistance in the diagnosis of syphilitic affections of the central 
nervous system. Fortunately it is possible to employ much larger 
quantities than is the case with blood serum because of the fact that 
the anticomplementary power of the fluid is light. By the use of 
graduated amounts, the reaction is placed upon a quantitative basis 




Fig. 120. — Meningococci in pus. X 1000. (Kendall.) 

which is helpful in following the progress of treated cases. The technic 
for the Wassermann reaction with spinal fluid has been given elsewhere 
(see page 151). 

Bacteriological Examination of the Spinal Fluid.— The fluid should 
have been drawn into a sterile test-tube. W T hen a bacterial infection 
is suspected, it is wise to remove a small quantity of the fluid with a 
sterile pipette and place it in a separate tube. This fraction can be 
used for the enumeration of the cells, for the globulin test, and to make 
slides for differential count and preliminary search for bacteria. 
Cultures should be made upon appropriate media with some of the 
remainder. A considerable portion of the fluid should then be placed 
in the incubator and kept at 37° C. for twenty-four to forty-eight hours. 
The fluid itself serves as an excellent culture medium. Cultures may 
be made from this, since the organisms multiply in the fluid, and cover- 
slips may be prepared for staining. Organisms are often found in this 
incubated fluid when none can be found in the fresh fluid. 



380 EXAMINATION OF BODY FLUIDS AXD EXUDATES 

Infection with Micrococcus Intraccllnlaris Meningitidis. — Usually, the 
fluid is turbid and even purulent. At times it may be so thick that it- 
will scarcely run through a wide-bore needle. The organism is a 
biscuit-shaped diplococcus, usually occurring in pairs but occasionally 
in fours or even in small masses. In form it resembles the gonococcus. 
It may be found both within and without the cells, and it is thought 
that the prognosis is better when it is intracellular (Fig. 120). It 
stains well with ordinary stains and is Gram-negative. Well-defined 
capsules are not demonstrable. The findings of a Gram-negative diplo- 
coccus in turbid spinal fluid containing a preponderance of polynuclear 
cells generally means infection with the meningococcus, although it is 
well to confirm this with cultures. 

Occasionally clear or almost clear spinal fluids show on microscopic 
examination polymorphonuclear and mononuclear cells in almost equal 
numbers and no organisms. Such fluids should be placed in the 
incubator, since often organisms are found in large number on the 
following day. 

For cultures, a suitable medium is glucose-ascitic agar, made with a 
meat infusion basis and with reaction neutral to phenolphthalein. In 
emergencies, Loeffler's blood serum may be used, or a few drops of 
whole human blood, obtained under sterile conditions, may be added to 
glucose agar which has been melted and whose temperature has been 
reduced to 40° C . This medium is poured into a sterile Petri dish and 
allowed to solidify. Some of the spinal fluid is streaked across the 
plate with a platinum loop when the fluid is fairly turbid, while a larger 
quantity should be run over the surfaces of the plate from a sterile 
pipette when the fluid is clear. The colonies are rather minute, grayish- 
white in color, and not confluent. 

Infection with Diplococcus Pneumoniae. — This is a more frequent 
complication of pneumonia than is generally recognized. The spinal 
fluid is usually turbid, occasionally purulent. The cells are increased 
in number, polymorphonuclear leukocytes predominating. The mor- 
phology and staining reactions of the organism have been described 
elsewhere (see page 360). In general terms, a Gram-positive, encapsu- 
lated lance-shaped diplococcus, in all reasonable probabilities can be 
only a pneumococcus. Confirmation may be made by cultural methods 
as described in the chapter on Sputum (see page 362). The fluid may 
be centrifugalized if desired and the sediment taken up with 1 cc. of 
sterile normal salt solution after decanting the supernatant fluid. This 
may be injected into a mouse for the purpose of determining the type 
of the organism. Dextrose blood broth may be inoculated with spinal 
fluid, and the cultures used for typing according to Avery's method. 

Infection with B. I n fin e?izoe. —The influenza bacillus is occasionally 
found in the spinal fluid, usually following infection elsewhere. Its 
morphology and staining have been described (see page 359). Cul- 
tures may be made by planting fairly large quantities, 1 or 2 cc, on 
blood-agar plates. 



EXAMINATION OF SPINAL FLUID 



381 



Infection with B. Tuberculosis.— When tuberculous infection is 
suspected, it is usually possible to secure a fairly large quantity of 
fluid. A part should be poured or pipetted off and used for the cell- 
count, globulin test, and Lange reaction. Another portion should be 
centrifugalized at a rapid rate. The supernatant fluid should be poured 
or pipetted off. The sediment is mixed with a drop or two of the fluid 
in the centrifuge tube, spread upon a cover-slip with 
the platinum loop, dried carefully, fixed by covering 
with absolute methyl alcohol for two minutes, and 
stained by the Ziehl-Neelsen method (see page 436) . 

The remainder of the fluid is allowed to stand 
in the incubator at 37° C. for about twenty-four 
hours '. A fibrin clot usually forms and attaches 
itself to the sides and bottom of the tube, entangling 
the tubercle bacilli in its meshes (Fig. 121). A 
clot is withdrawn with a sterile platinum wire, is 
teased out on a slide, dried, fixed with heat, and 
stained for tubercle bacilli in the usual manner. 

A variation in technic which gives excellent 
results is to pour the spinal fluid into a short 
sterile graduate or into a small sterile Stender dish. 
The vessel must be large enough to permit a cover- 
slip to rest flat in the bottom. The fluid is allowed 
to stand in the incubator for about twenty-four 
hours. The cover-slip is then removed from the 
bottom of the container by lifting it up with a 
platinum wire bent to form a wide loop. This loop 
is slipped under the cover-slip and the cover-slip is 
raised without tilting it, keeping it in a horizontal 
plane. The fibrin clot will adhere to its upper 
surface, and will not require teasing out. After 
drying and fixing, it may be stained in the usual 
way. 

Infection with Other Organisms.— Other organisms 
which may cause meningitis are the streptococci, 
the staphylococci, the colon bacillus, the typhoid 
bacillus, the Bacillus pyocyaneus, Micrococcus tetra- 
genous, yeasts, etc. In almost all instances, menin- 
gitis due to these invasions by organisms is second- 
ary to infection elsewhere, such as otitis media, 
mastoid infection, infections of the nasal cavities or accessory sinuses, 
wounds of skull or scalp, endocarditis, erysipelas, etc. 

Significance of the Findings.— Cell-count.— Increased cell-count is 
found in any active meningitis. In making a differential count of the 
cells, the polynuclears will be found to predominate in the infections 
due to the pneumococcus, the meningococcus, and the pyogenic 



.Fig. 121.— Fibrin 
clot in spinal fluid in 
tuberculosis. 



382 EXAMINATION OF BODY FLUIDS AND EXUDATES 

organisms, while in tuberculosis the lymphocytes comprise 90 to 95 
per cent, of the cells. This rule, however, is not a rigid one. For 
example, when the infection with meningococcus has passed into a 
subacute stage, the polymorphonuclear cells may be equaled or 
exceeded in number by the lymphocytes". In syphilitic infections, the 
cells are practically all small round cells of lymphocyte type. 

Qlobulin.— Increased globulin is due to transudation from vessels of 
the central nervous system. It may be seen in tumors of cord or brain, 
of bony canal or of meninges, in arteriosclerosis, or any inflammatory 
condition of the central nervous system. 

Wa8sermann Reactions.— A positive reaction is found only in the 
spinal fluid when syphilitic involvement of the central nervous system 
is present. 

Lange CoUoidalr-gold Curve. — The findings have been given in the 
preceding paragraphs describing the reaction. 

Characteristic Findings (Tuberculous Meningitis.)— The fluid is 
usually absolutely clear and under increased pressure, globulin is 
increased, and cell-count is high. A differential cell-count usually 
shows predominance of lymphocytes, though this is not invariable, for 
polymorphonuclears are occasionally formed in the majority, especially 
in the early stages. Tubercle bacilli may be found. 

Syphilitic Infections (paresis, tabes dorsalis, syphilitic meningo- 
endarteritis).— The blood Wassermann reaction is often positive. This 
is not constant and a negative Wassermann reaction in the blood 
does not exclude syphilis of the nervous system. The spinal fluid is 
usually clear. It may be under slightly increased pressure. Usually 
the Wassermann reaction in the spinal fluid is positive, cell-count 
increased and globulin in excess of normal. The Lange colloidal-gold 
curve is helpful in differentiating between these conditions. 

Acute Anterior Poliomyelitis. —The fluid may be clear, slightly hazy, 
or even flocculent. The cells are usually increased, the count ranging 
from to 2000 per c.mm. The globulin may be greatly increased. 
The differential count usually shows above 50 per cent, polymorpho- 
nuclears in the early stage, while later there are usually 90 per cent, 
or more mononuclears. In general, characteristics of the fluid depend 
to some extent upon the severity of the process. 

Other Injections.— The fluid is usually purulent. With any acute 
infection, the globulin is increased and the cell count high. The 
differential count usually shows a vast majority (90 per cent, or over) 
of polymorphonuclear cells. When the infection becomes chronic, the 
mononuclears often become more numerous, and may predominate 
over the polynucleiirs. The determination of the form of meningitis 
rests upon detection of the inciting organism. The more frequent 
invaders are the Micrococcus intracellularis meningitidis and the 
pneumococcus. 



EXAMINATION OF THORACIC AND ABDOMINAL FLUIDS 383 

EXAMINATION OF THORACIC AND ABDOMINAL FLUIDS. 

Technic of Obtaining Fluid.— The point for exploratory puncture of 
chest or abdomen should be determined only after careful physical 
examination, and should be below the upper level of the area of dullness. 
For paracentesis thoracis (thoracentesis) the patient should be propped 
up in bed on pillows or a back-rest. If the point chosen be in the sixth 
or seventh interspace, it is kept in the midaxillary line, while if it be in 
the eighth interspace, it should be below the angle of the scapula. 
Paracentesis abdominis is usually made in the median line 5 to 8 cm. 
above the upper border of the pubic bones. The bladder should be 
completely emptied, by catheterization if need be, and the patient 
should sit upright on the side of the bed. 

It should be remembered that puncture of the chest is not free from 
danger, particularly when large quantities of fluid are removed, and 
the operator should be ready for emergencies. Acute pulmonary 
edema may occur. Often it may be relieved by the hypodermic injec- 
tion of morphine and atropine. When these failed, Riesman found dry 
cupping efficacious. 

The skin over the site of puncture should be thoroughly sterilized 
by scrubbing with green soap, ether, alcohol, and finally by the applica- 
tion of tincture of iodine. Surgical asepsis should be observed with 
regard to the preparation of the operator's hands and the instruments. 
Local anesthesia may be effected by infiltrating the skin and subcuta- 
neous tissues with a sterile 1 per cent, solution of novocaine, using a fine 
hypodermic needle fitted to a glass hypodermic syringe. The needle 
selected for puncture should be of much larger bore than the ordinary 
hypodermic needle, since a fine needle would be easily occluded with 
bits of tissue or clotted fibrin. One of about 18 gauge, fitted with a 
removable obturator or mandarin, is suitable. If a small quantity is 
desired for diagnostic purposes only, a glass syringe of the Luer type 
may be used for aspiration, but if the withdrawal of a large quantity of 
fluid be indicated, the needle should be one which can be attached to 
the Potain aspirator. 

Examination should be made as soon as possible after its removal. 
The routine examination should include the following: (1) Observa- 
tion of gross characteristics— color, odor, turbidity, presence of blood. 
(2) determination of specific gravity. (3) determination of amount of 
albumin. (4) cytological study. (5) bacteriological study. (6) (a) 
cover-slip preparations, (b) cultures when indicated by other findings 
and clinical features, (c) search for tubercle bacillus. 

Gross Characteristics.— Transudates are effusions due to disturbances 
of circulation or to changes in the osmotic relations between blood 
plasma and body tissues. They are usually transparent, light yellow 
fluids. Exudates, which are the result of inflammatory processes, 
show considerable variation in appearance, ranging from serous fluids 
to definitely purulent ones. Chylous exudates are white and milky 



384 EXAMINATION OF BODY FLUIDS AXD EXUDATES 

due to the presence of large amounts of fat. They are seen after the 
rupture of a lymphatic trunk into the pleura or peritoneum. Chyloid 
exudates are seen in malignant involvement of the pleura or peritoneum. 
They are somewhat milky in appearance though not so much so as are 
the chylous exudates. The opacity is due partly to fat, largely to 
degenerated and broken-down cells, and sometimes to lecithin. Hemor- 
rhagic exudates are seen after injuries to the chest wall, in the presence 
of new growths, and when the pleura is involved in a tuberculous 
process. At times the presence of blood in exudates and transudates 
is due to trauma inflicted by the exploratory needle. Purulent exudates 
are thicker and yellow to green in color. Leukocytes are present in 
large numbers; bacteria may be found, and occasionally, crystals. of 
fatty acids, some fat and at times, crystals of cholesterol. 

Specific Gravity.— This may be taken at room temperature with an 
urinary hydrometer. The specific gravity of ascitic fluids and pleural 
transudates is usually below 1.015 while that of exudates is above 
1.018. 

Determination of Amount of Albumin.— The fluid should be diluted 
(adding to one part of fluid 9 parts of distilled water) and thoroughly 
acidified to litmus with 5 per cent, acetic acid. The Esbach test is 
carried out as directed in the chapter on Urine (see page 242), multiply- 
ing the reading by 10. 

Pleural transudates contain less than 25 gm. per liter; peritoneal 
transudates from 15 to 20 gm. per liter; while pleural exudates contain 
from 30 to 65 gm. and peritoneal exudates from 20 to 25 gm. per liter. 

Cytological Examination. 1 — Preparation of Sediment.— The fluid should 
be examined as soon as possible after its withdrawal. If clotted, 
the fluid should be shaken in a sterile bottle with small sterile glass 
beads or stirred with a sterile glass rod in order to cause contraction of 
the clot and liberation of the entangled cells. The fluid is centrifugal- 
ized in a 15 cc. sterile centrifuge tube at a rapid rate (2500 to 3000 
revolutions per minute) for from three to five minutes. The supernatant 
fluid is decanted carefully without disturbing the sediment, which 
with a platinum loop is mixed with the few remaining drops of fluid 
and spread upon a slide in constantly increasing concentric circles. 
The preparation is allowed to dry in the air without the use of heat to 
avoid distortion of the cell outlines. Treatment with a separate 
fixative is not necessary, since Wright's stain contains methyl alcohol. 
Should cover-slips be prepared for bacteriologic" examination with 
other stains, they may be fixed by covering with absolute methyl 
alcohol for two minutes. 

Staining. — Musgrave recommends staining with the following: 

Wright's blood stain 3 parts 

Absolute methyl alcohol 1 part 

The cover-slip is flooded with the stain, which is allowed to remain 
for a minute or slightly less, when S to 10 drops of water are added as 

1 For a discussion of the cytology of serous effusions, see articles by Percy Musgrave. 



EXAMINATION OF THORACIC AND ABDOMINAL FLUIDS 385 

in staining blood preparations. The stain, so diluted, is allowed to 
remain an additional two or three minutes, when it is poured off and 
the cover-slip flooded several times with water from a medicine dropper 
and dried by holding between the thumb and forefinger and whisking 
through the Bunsen flame. It may be mounted in balsam. 

Findings.— Red blood cells, white blood cells, and endothelial cells 
may be seen. A differential count should be made of the white cells 
to determine the proportion of the different forms. Those ordinarily 
encountered are the lymphocytes, polymorphonuclear neutrophilic cells, 
and occasionally eosinophilic polymorphonuclears. Large numbers of 
broken-down cells may be seen. The endothelial cells are cast off from 
the serous surfaces and are of relatively large size. They may be seen 
as isolated cells or in groups of two or more. These cells are usually 
circular in outline with round or oblong nuclei. They are seen 
in various states of degeneration, sometimes with disintegrated and 
ragged protoplasm, and sometimes with the protoplasm swollen and 
more or less vacuolated. They are phagocytic at times, containing 
partly digested leukocytes. Cancer cells may be seen. They are 
larger than the endothelial cells. Identification, which is not always 
easy, may be aided by the discovery of nuclear division. 

Interpretation of Cytological Findings.— So-called laws regarding the 
cytology of serous fluids were formulated by Widal and Ravaut. 
Briefly, these may be summarized as follows: 

1. In a tuberculous process the lymphocytes predominate. 

2. In an acute infectious process the polymorphonuclear cells 
predominate. 

3. With a mechanical effusion or transudate, endothelial cells are 
seen in large numbers. They are especially likely to occur in large 
sheets or plaques. 

There are, however, conspicuous and. frequent exceptions to these 
so-called laws. In the early stages of a tuberculous process, poly- 
morphonuclear cells often predominate, while lymphocytosis is some- 
times seen in transudates and in infections with staphylococcus, 
influenza bacillus and pneumococcus. Particularly is this the case in 
chronic infections. 

Bacteriologic Examination.— All purulent fluids and such serous fluids 
as are not definitely transudates, should be subjected to bacterio- 
logical study. Cover-slip preparations may be made of serous fluids 
as described for cytological study and microscopic examination for 
bacteria. A preliminary search may be made in the cover-slip stained 
with Wright's stain, but a Gram's stain should be carried out also. 
When influenza is suspected, a cover-slip should be stained with dilute 
carbol-fuchsin. In chest fluids the same varieties of organisms may be 
expected as are found in the sputum, and the methods given in the 
chapter on Sputum for staining and cultural work can be adapted to 
the examination of pleural fluids. 

If the pneumococcus be present in empyema fluid, suitable media 
25 



386 EXAMINATION OF BODY FLUIDS AND EXUDATES 

should be inoculated as directed in the chapter on Sputum (see page 
362) and the type determined by the agglutination tests. If desired 
as an additional measure, a portion of the fluid may be centrifugalized 
at high speed to throw down the pneumococci when the sediment may 
be taken up with 1 cc. of sterile normal salt solution and injected 
intraperitoneally into a white mouse, or used for cultural methods. 

Influenza bacillus may be cultivated on blood agar, blood oleate 
agar, or in blood oleate broth (see page 359). 

The detection of the tubercle bacillus in the pleural fluid is often a 
matter of the utmost importance. A cover-slip may be prepared as 
directed for the cytological study, fixed with absolute methyl alcohol, 
and stained for tubercle bacillus (see page 436). A search conducted 
in this way, however, is seldom successful. 

Inoscopy is the name given by Jousset to a method which he pro- 
posed for the detection of the tubercle bacillus. The clot is removed 
with a sterile wire, placed on a piece of sterile gauze which has been 
tied across the top of a sterile glass funnel and freed from serum by 
pouring sterile freshly distilled water through the gauze. It is then 
picked up with a sterile spatula and placed in a small bottle with about 
20 or 30 cc. of the following mixture: 

Pepsin 2 gm. 

Strong hydrochloric acid 10 cc. 

Pure glycerin 10 " 

Sodium fluoride 3 gm. 

Distilled water 1000 cc. 

The bottle containing the clot with the pepsin mixture is placed in 
the incubator at 37° C. or in a water-bath at 50° C. until digestion has 
been accomplished. The mixture is then centrifugalized. Cover-slip 
preparations are made as directed for cytological study and stained 
for tubercle bacilli. Musgrave states that the majority of the organ- 
isms which he found with this method were broader and shorter and 
some were paler red than those usually found in the sputum. 

Animal inoculation is probably the most reliable method for demon- 
strating the organisms, though the results are delayed. The fluid 
may be concentrated by centrifugalization (see page 401 for method). 

EXAMINATION OF PURULENT EXUDATES. 

Pus is usually thick, and yellow or grayish-yellow in color. It 
contains large numbers of cells, chiefly polymorphonuclear neutrophiles 
("pus cells") in various stages of degeneration. The cell outlines are 
often vague and in many instances nuclei and cytoplasm are poorly 
stained. Ingested bacteria may be seen within the leukocytes. In 
gonorrheal pus, eosinophiles are seen. 

Pus should be obtained with a sterile swab, platinum loop, or capil- 
lary pipette. Smears should be made on slides or cover-slips and stained 
,it once (see page 414 for method). Preliminary examinations may 
render Identification possible on morphology and staining reactions, 



EXAMINATION OF PURULENT EXUDATES 



387 



and at least serve to indicate the type of media which should be 
employed for cultures. 

For routine purposes, Loeffler's methylene blue and Gram's stain 
should be employed. Gram's stain is most useful in differential 
diagnosis (see page 436) . 




Fig. 122.— Staphylococcus. X 1100 diameters. (Park and Williams.) 

Among the organisms which are more frequently found in patho- 
logical exudates are staphylococci, streptococci, pneumococci, and 
gonococci. Organisms of the staphylococcus group are characterized 
by the occurrence in cultures of irregular grape-like clusters (Fig. 122). 
The cocci are spherical, averaging about 0.8^ in diameter, and are 
Gram-positive. Cultures are necessary for the differentiation of the 
varieties. 




Fig. 123.— Streptococcus in pus. X 800. (Kendall.) 

Streptococci may be seen in pus in chains (Fig. 123), but differentia- 
tion is not always possible since they may occur as separate cocci and 
only show chain formation when grown in liquid media. The individ- 
ual cocci are from 0.5 to 1/j, in diameter. While ordinarily spherical, 
adjacent cocci in chains may show some flattening of the opposed 
surfaces which are in apposition. Practically all of the streptococci are 
Gram-positive, Cultures are necessary for the determination of species. 



388 EXAMINATION OF BODY FLUIDS AND EXUDATES 

Pmumococci may be seen, especially in pus from pleural exudates. 
When lance-shaped Gram-positive diplococci are seen, preparations 
should be stained with one of the capsule stains (see Bacteriological 
Methods). A description of the organism with methods for cultiva- 
tion and typing, may be found in the chapter on Examination of 
Sputum. 

The Eye.— Pus from the conjunctiva may show staphylococci, strep- 
tococci, pneumococci, diphtheria bacilli and gonococci. The prompt 
recognition of the latter (see page 393) is a matter of great importance, 
on account of the rapid and destructive course of the gonorrheal 
ophthalmia. The pneumococcus is occasionally found associated with 
a corneal ulcer. The Koch-Weeks' bacillus is found in acute epidemic 
conjunctivitis. It is Gram-negative and similar in morphology to B. 
influenza, but somewhat longer (Fig. 124). It grows readily upon 
media containing serum or ascitic fluid and does not require the 




Fig. 124. — Koch- Weeks' bacillus from "pink-eye" — third generation. 
X 1000 diameters. (Weeks.) 

presence of hemoglobin in the medium as does the influenza bacillus. 
The Morax-Axenfeld bacillus is associated with certain cases of sub- 
acute and chronic conjunctivitis in which the inflammation is marked 
in the angles, particularly about the caruncle. The Gram-negative 
organisms are short and thick, usually about 2/x long, with distinctly 
rounded ends, often in short chains. They stain readily with the 
ordinary stains. They grow only upon alkaline media containing blood 
or blood serum and when cultivated upon Loeffler's blood serum, 
cause liquefaction of the medium. The diphtheria bacillus may be 
found in membranous conjunctivitis (Figs. 127 and 128). In examin- 
ing smears from the conjunctiva, it must be remembered that the 
Bacillus xerosis is found in normal conjunctivas and that it cannot be 
distinguished from the diphtheria bacillus on morphological grounds. 
Growth upon Loeffler's blood serum is very similar, too, differing only 
in being a little more delicate. Differentiation can be made only by 
the use of sugar media. B. xerosis forms acid from saccharose and not 



EXAMINATION OF PURULENT EXUDATES 389 

from dextrin while B. diphtherias forms acid from dextrin and not from 
saccharose. The trachoma bodies of Halberstadter and Prowazek may 
be demonstrated by examining scrapings from the conjunctiva. They 
are small oval or round granules of variable size, typically enclosed in a 
"mantle;" found within the bodies of the conjunctival epithelial cells, 
usually near the cell-nucleus. With earlier lesions, the bodies stain a 
faint blue with Giemsa's stain, while later they stain red. Williams 
believes that these bodies are degenerated hemoglobinophilic bacilli. 

The Ear.— The more frequent causes of acute otitis media are the 
pneumococcus, the streptococcus, and the diphtheria bacillus. Dis- 
charges may occur secondary to other infections, such as typhoid fever 
or influenza and the exciting cause of the original infection will be found 
in the discharge. As an otitis becomes chronic, contamination with 
the staphylococcus may take place. B. pyocyaneus is sometimes 
found, as is the colon bacillus and Friedlander's bacillus. 

The Mouth and Throat.— A large variety of organisms may be found 
in the mouth. The number depends somewhat upon the care which 
is given to the teeth. Even in normal individuals, many are always 
present, including staphylococci, streptococci, occasionally pneumo- 
cocci, Leptothrix buccalis, and Spirocheta buccalis and Spirocheta 
dentium. The spirochetes are found in especially large numbers when 
the teeth and gums are in poor condition. Leptothrix buccalis occurs 
as long, slender- jointed threads. Spirocheta dentium is about 4 to 10^ 
long, is delicate and has deep curves, so that it is easily mistaken for 
Treponema pallidum. Spirocheta buccalis is longer, thicker, and has 
coarser curves and resembles Spirocheta vincenti. Endamaba gingi- 
valis (E. buccalis) is found about the teeth. It may be found in the 
pus obtained from the sockets of the teeth in cases of pyorrhea alveolaris 
and has been regarded by some as a causative factor. Ameboid move- 
ment may be seen when material is examined at once on a warm stage. 
The size varies from 6 to 32^. Ectoplasm is present, the nucleus 
vesicular and poor in chromatin, and ingested red blood cells, leuko- 
cytes, and bacteria are seen. Bass and Johns have suggested the 
following method of staining. Pus is obtained from pockets about 
the teeth with a Younger dental scaler No. 22 or No. 23. It may be 
examined in the moist state on a warm slide, or stained according to the 
technic of Bass and Johns. The pus is spread to make a thin film, 
dried in the air, fixed by passing through the flame 2 or 3 times, and 
covered with a drop or two of carbol-fuchsin. This is washed off 
immediately with water and replaced with a drop or two of Loeffler's 
methylene blue, which is allowed to act for about one-half minute. 
The slide is washed off with water and dried. 

Thrush.— When scrapings are taken from the white patches on the 
surface of the mucous membrane and squeezed out on a slide, a net- 
work of O'idium albicans will be seen, containing budding cells which 
resemble yeast cells and under suitable conditions show mycelia and 
spores. There are two varieties, one with small spores which does not 



390 



EX AM IX AT I OX OF BODY FLUIDS AXD EXUDATES 



liquefy gelatine and one with large spores which docs liquefy gelatine. 
Sugars are fermented. 

Pseudomembranous lesions of the mouth, tonsils, and pharynx arc of 
great importance from a clinical standpoint. Given a membranous 
lesion, the principle possibilities are diphtheria, Vincent's angina, and 
syphilis. 

Syphilitic Lesions. — X primary lesion of the mouth or tonsil is 
occasionally seen. There is usually much induration of the deeper 
tissue. The Treponema pallidum (Fig. 125) may be found though 
there is some possibility of confusion on account of the presence of 
Spirocheta dentium in the mouth. In the secondary stage, the patches 
are usually flat, and do not show a distinct membrane, only a filmy, 
gray surface. The likelihood of confusion with diphtheria or Vincent's 
angina is slight on account of the duration of the lesions, their number, 
and appearance. In this stage the Tr. pallidum may be demonstrated 





-Treponema pallidum. 
(Kendall.) 



Fig. 126. — Vincent's angina. Bacillus 
and Spirillum fusiformis. (Kendall.) 



and the Wassermann reaction is almost invariably positive. In the 
so-called , tertiary stage, there may be lesions of varying size, often- 
times of great extent, showing marked loss of tissue, and covered at 
times with a dirty gray or yellow pseudomembrane. The history of 
chronicity, admission by the patient of infection and a positive Wasser- 
mann reaction will aid the diagnosis. Tr. pallidum is rarely found in 
the tertiary stage. 

In Vincent's angina, the lesion usually involves the tonsils and often 
the soft palate. A punched-out ulcer results and tissue destruction 
may cause the loss of tonsils, uvula, and portions of the palate. The 
ulcerated area is covered with an adherent dirty gray or yellow mem- 
brane which is detached with some difficulty. After cleaning the 
surface by gentle swabbing, portions may be removed for examination 
with a dull curette, placed on a slide, and squeezed with the aid of a 
second slide to make a thin preparation. A clear picture is given by 



EXAMINATION OF PURULENT EXUDATES 391 

staining for five minutes with diluted carbol-fuohsin (one part of carbol- 
fuchsin and 9 parts of distilled water). Two organisms arc seen, the 
Spirocheta vincenti and 13. fnsiformis. The spirochetes are motile, 
rather slender, feebly staining organisms, abont 10 to 20 fi long, and 
irregularly curved with shallow undulations. The fusiform bacillus 
is from 3 to 10;u long, and thicker at the center than at the end, which 
taper to a point. The rods stain rather unevenly so that they appear 
to be barred (Fig. 126). The bacillus is not motile, though Stitt states 
that he has observed feeble motility in specimens mounted in saliva. 
Both organisms are Gram-negative, though there is a tendency to some 
variation on the part of the bacillus. It should be remembered that 
these organisms may be secondary invaders, and that they are fre- 
quently present in great numbers in syphilitic lesions. 

In diphtheria, the membranous exudate is usually seen in the naso- 
pharynx, on the tonsil, and less frequently in the nose and trachea. 
Local treatment should be withheld for twelve hours previous to making 
cultures. In making cultures from children, the wire bearing the 
cotton swab should be curved like a laryngeal applicator and intro- 
duced deep into the throat. Inoculation should be made on Loeffler's 
blood-serum; then the swab should be rubbed on slides and the resulting 
smear stained immediately. 

Cultures are incubated at 37° C. for not over sixteen or eighteen 
hours, when some of the growth should be removed with a sterile plati- 
num loop and transferred to a slide. A thin emulsion should be made 
with a droplet of sterile water. This is dried, fixed in the usual way, 
and stained by one of the following methods: 

1. Stain with Loeffler's methylene blue for five minutes, using no 
heat. Pour off the stain and wash with repeated changes of 0.5 per 
cent, acetic acid until the preparation assumes a gray tint, showing 
only a blue tinge. This method has the advantage of simplicity and 
gives a very clear-cut picture. The polar granules are dark blue, 
standing out clearly in contrast in the very pale bodies of the bacilli. 

2. Stain with Neisser's stain for from one to three seconds. (See 
page 436.) Wash in water and stain for from three to five seconds 
with Bismarck-brown solution. Wash with water and examine with oil- 
immersion lens. With this method the bacilli are seen as brown rods 
containing bluish-black granules. 

Diphtheria bacilli are straight or slightly curved rods, staining 
irregularly, showing metachromatic granules which are spoken of as 
"polar granules" when situated at the ends of the rods. Barred forms 
appear (Figs. 127 and 128). A tendency to parallel arrangement is 
seen. Oftentimes the rods are quite irregular and clubbed ends are 
seen. The morphology of the organism varies at different periods of 
development. This is to be remembered in examining cultures, and 
makes the time of incubation a matter of practical importance. Vari- 
ous workers have divided the organism in groups based on the mor- 
phology. 



392 EXAMINATION OF BODY FLUIDS AND EXUDATES 

There is no way in which virulent strains can be distinguished from 
non-virulent ones on morphological grounds. One cc. of a forty-eight- 
hour broth culture may be injected intraperitoneally into each of 
two guinea-pigs. If the culture be virulent, death should occur in 
one to two days. At the point of inoculation there will be a sero- 
sanguineous exudate. 





Fig. 127. — Bacillus diphtherise, methy- 
lene blue stain. X 1000. (Kendall.) 



Fig. 128. — Bacillus diphtherise, branch- 
ing. X 800. (Kendall.) 



The Nose.— Cultures from the nose are of importance at times. 
They may be made by introducing a swab into the nares. A speculum 
may be used but it is not usually necessary. For cultures from the 
retropharyngeal space, a special tube has been devised by West. 
This consists of a glass tube about 7 mm. inside diameter and about 
16.5 cc. long, curved at one end nearly to a right angle. Inside the 
glass tube is a copper wire carrying at one end a cotton swab. The 
swab is pulled back into the tube, both ends of the tube are plugged, 
and the apparatus is sterilized. When cultures are to be made, the 
glass tube is carried back over the tongue as near to the posterior 
pharyngeal wall as ispossible. The copper wire ispushed forward enough 
to permit swabbing of the mucous membrane, when it is pulled back 
into the glass carrier and removed from the mouth. The plate upon 
which cultures are to be made is then swabbed with the material 
which has been obtained. Many workers prefer simply a curved wire 
the distal end of which has been wrapped with cotton to form a swab. 

Nasal cultures assume especial importance in the detection of 
carriers of meningococci, streptococci, and diphtheria bacilli. In 
attempting to determine carriers, suitable media should be used, for 
example, blood-agar plates for the streptococci, and glucose agar 
(0.4 + to phenolphthalein) with either blood, blood serum, or ascitic 
fluid for meningococci, and Loeffler's blood serum for diphtheria bacilli. 

Urethra and Vagina.— Pus is obtained with a sterile swab. With 
the adult female, a specimen should be taken from the mouth of the 



SKIN INFECTIONS 393 

urethra, and another from the mouth of the cervix with the aid of a 
speculum. The material is spread upon the slide gently to avoid 
injuring the cells and fixed and stained with Grain's stain and methy- 
lene blue according to the methods described in the chapter on bacterio- 
logical technic (see pages 435 and 436) . 

Gonococci are seen as Gram-negative, flattened, biscuit-shaped 
spheres, occurring in pairs with flattened sides in apposition. The 
cocci are about O.Sju wide by about 1.6m long. Usually a number are 
clustered together, and in acute cases many are found within the 
cytoplasm of the leukocytes (Fig. 129). They do not grow well on 
ordinary culture media, requiring the presence in medium of blood 
serum, ascitic or hydrocele fluid. In pus from the male urethra the 
occurrence of organisms with the'morphology and staining reactions 




Fig. 129. — Gonococcus smear of pus from acute case. Methylene blue stain. (Warden.) 

just described is ordinarily taken as sufficient for the purpose of 
practical diagnosis. An organism which may occur in the urethra 
and which may cause confusion is Micrococcus catarrhalis, which is 
also a Gram-negative diplococcus. In contrast to the gonococcus 
this grows readily upon ordinary media. "Shreds" may be found in 
the urine in acute and chronic gonorrheal infections (see page 208). 

SKIN INFECTIONS. 

A number of chronic infections of the skin are caused by the patho- 
genic moulds. Examination of scrapings is of diagnostic assistance. 
The crust, skin, or an infected hair is removed and softened with a drop 
or two of 20 per cent, sodium hydrate, is pressed out on a slide with a 



394 SKIN INFECTIONS 

cover-slip, when the slide may be heated slightly over the flame. Stain- 
ing is usually unnecessary, but may be accomplished by placing some 
of the material on a slide. It is smeared with a drop of serum or 
Mayer's egg-albumen and treated with alcohol and then with ether to 
free it from fat. It may be stained with Wright's stain or by Gram's 
method. 

Achorion Schoenleinii.— Fa vus is a chronic disease, usually attacking 
the hairy portions of the body. Small yellow sulphur-like cupped-out 
crusts are seen surrounding a hair or hairs. When removed and 
treated with sodium hydrate, the microscopic examination shows that 
the crust (scutulum) is made up of desquamated epithelial cells, dried 
serum, and double-contoured spores of irregular shape, lying in a 
net-work of mycelial threads, the fungus being known as Achorion 
, Schoenleinii. It is possible at times to see filaments of mycelia which 
give off hypha? terminating in small knobs. Cultures may be obtained 
by grinding some of the material in a sterile mortar with fine sterile 
sand. The triturated mass may be inoculated into melted agar and 
plates poured. Growth is best, however, on the surface of Sabouraud's 
glucose or maltose agar and appears in from six to nine days in the form 
of a pale yellow whorl the surface of which takes on a convoluted appear- 
ance as the colony increases in size. To inoculate, the tip of a curved 
pointed platinum needle is moistened on the sterile medium. A small 
particle of the scutulum is picked up on the moistened end and deposited 
on the medium. Several different plantings should be made on each tube. 
In making cultures of hairs in a case of favus, it is not necessary to 
choose any particular hair. A hair may be removed from the scalp, 
cut into small pieces with a sterile knife blade on a sterile glass slide 
and planted on the medium, using the same technic as for favus 
cultures. 

Ringworm.— Ringworm of the hairy portions of the body (Tinea 
tonsurans, T. barbae, or Tinea sycosis) and of the hairless portions of 
the skin (Tinea circinata) is caused by several varieties of trichophyton 
which belong to a group of fungi imperfecti. There are two types of 
fungus, those with small spores (about 2 to 3/x in diameter) (Trichoph- 
yton microsporon) and those with larger spores (about 7 to 8/x in 
diameter) (Trichophyton megalosporon). The small-spored variety is 
found throughout the hair substance, from which branches pass out 
to the surface. Ectospores are found on the surface in zooglea-like 
masses. The mycelium is difficult to see. The microsporon is the 
common fungus in Trichophytosis capitis as seen in iimerica and 
England. With Trichophyton megalosporon endothrix the mycelium 
occupies the center of the hair-shaft, running its entire length while 
with Trichophyton megalosporon ectothrix, the fungus is found in the 
intrafoUicular region and forms a sheath between the hair and the 
follicle. The fungi are seen as finely interlaced separate threads upon 
which swellings appear. From these the chlamydospores develop. 
Ringworm of the body may be caused by either the small spore or large 
spore organism. 



SKIN INFECTIONS 395 

These fungi are best cultivated upon media enriched with glucose 
or maltose. Infected hairs or scrapings of skin may be treated for two 
hours with 60 per cent, alcohol to kill the bacteria before inoculation 
and then planted according to the technic given for favus cultures. 
In this condition, in distinction from favus, all the hairs are not affected 
and one must select the broken off stubs which show just above the 
surface of the skin and which on removal are seen to have a glove-like 
whitish deposit over the outside, i. e., a fungus growth. Sabouraud's 
medium is generally recommended: 1 

Maltose or glucose 4.0 gm. 

Peptone 1.0" 

Agar 1.8" 

Water 100.0 cc. 

Trichophytosis Unguis.— In making cultures from ringworms of the 
nails the same technic may be employed as with the cultures of the 
hairs, cutting the pieces finely. For microscopic examination, it is 
often necessary to digest the broken off nails ten or twelve hours in a 
20 per cent, solution of sodium hydrate before making the examination. 

Microsporon Furfur.— This fungus is the cause of tinea versicolor 
(pityriasis versicolor), in which the skin shows dirty yellow or brown 
patches. The short, thick hyphse lie in a thick net- work and are 
usually not jointed or branched. The spores (conidia) may be seen at 
the extremities of the hyphse. The spore, whose diameter is a little 
greater than that of a red blood cell, contains a large nucleus. The 
fungus is cultivated with great difficulty. 

Oidium Albicans (Monilia albicans) .—In blastomycosis of the skin 
there is a nodular eruption which later forms a pustule. This discharge 
shows oval yeast bodies, about 12 to 14/x in diameter. Budding is seen. 
Cultivation is usually carried out easily by inoculating pus on Sabou- 
raud's glucose or maltose agar, which inhibits the growth of the ordinary 
cocci. Ormsby advises making cultures from the minute, deep-seated 
abscesses. 

Sporotrichosis.— In sporotrichosis of the skin there is a more or less 
nodular eruption which may or may not be generalized, and from which 
profuse yellow pus may be obtained. The process is chronic, tending 
to spread. Cultures from the pus on Sabouraud's media within four 
or five days show a beginning growth of dark brownish-black colonies 
which assume a more or less convoluted form and grow rapidly. The 
surface is quite characteristic, resembling the skin of a rattle-snake in 
color. 

Infestation with Itch Mite and Insects.— Sarcoptes Scabiei.— The itch 
mite is found on those portions of the body covered with thin skin, 

1 In culture work with ringworm, it is found that the characteristics vary with media 
from different sources. Hence, Sabouraud has recommended the use of the media first 
employed by him in his work, which is now used universally by men doing this work. 
The material can be procured from E. Cogit & Co., Boulevard St. Michael, Paris, 
France. The maltose and glucose, while in crude form, are much cheaper than the 
purified products and serve the purpose. In ordering it is well to ask for the prepara- 
tions used by Sabouraud in his work. 



396 DETECTION OF TREPONEMA PALLIDUM 

particularly between the fingers, and in the bend of the elbow- and knee- 
joints, in the inguinal and mammary region, and on the penis. The 
body is nearly circular in outline, marked by transverse striae on the 
back. There are four pairs of legs, two anterior and two posterior. 
The mite burrows under the skin in somewhat tortuous tunnels, which 
may be from a few millimeters to a centimeter in length. At the end 
of the tunnel the female may be found. Eggs and fecal material are 
deposited in the tunnels. The males are seldom found since they die 
after copulation. To assist in diagnosis, the tunnel may be opened 
with a needle and the contents pressed out upon a slide for examination. 

Pediculus Capitis.— The head louse lives in the hair of the scalp, 
rarely in the hair of other portions of the body. The color varies from 
light gray to black. The thorax and abdomen are of the same breadth, 
the former having six segments, the latter three segments and three 
pairs of legs. The male is from 1 to 1.5 mm. long and the female from 
1.8 to 2 mm. long. The ova (nits) are pear- or barrel-shaped and are 
attached to the shaft of the host's hair with a clasping collar. 

Pediculus Vestimenti.— The body louse lives on the clothing around 
the neck, throat and trunk. It is 2 to 4 mm. long and grayish-white in 
color. The abdomen is broader than the thorax and the antenna? are 
longer than those of the head louse. 

Pediculus Inguinalis.— The "crab-louse" is found in the hair of the 
inguinal region, rarely on the head, but occasionally on other parts of 
the body. The males are from 0.8 to 1 mm. long and the females about 
1.2 mm. long. The abdomen has nine segments. 

The diagnosis in cases of pediculosis may be made by finding the adult 
insects, but more readily by finding the eggs attached to the hairs in 
the affected regions. 



DETECTION OF TREPONEMA PALLIDUM IN SYPHILITIC 
LESIONS. 

In an early chancre, the organisms are usually demonstrated readily. 
After healing has commenced or after antiseptics or caustics have been 
used, the search may be fruitless. For this reason, local treatment 
should be withheld until a diagnosis has been made. The organisms 
may be found in any of the secondary lesions. In material obtained 
from the buccal cavity, there is always possibility of confusion on 
account of the presence of Spirocheta dentium. Treponemata are 
seldom found in serum obtained from tertiary lesions and rarely even 
in suitably stained sections of tissue. An essential part of the technic 
is obtaining "irritation serum." At times, serum wells out freely 
without irritation, but such cases are exceptional. The treponemata 
may be demonstrated in stained specimens, or in unstained specimens 
with the aid of dark-field illumination. The latter is distinctly the 
method of choice since movement may be studied as well as form, and 



OBTAINING IRRITATION SERUM FOR EXAMINATION 397 

the organisms are found much more readily. All staining methods are 
open to the objection that the apparent width of the organism varies 
with the thickness of the film. 



OBTAINING IRRITATION SERUM FOR EXAMINATION. 

The lesion should be thoroughly cleaned by repeated applications 
of sterile physiological salt solution and the surface scraped with a dull 
curette until the blood begins to ooze. Suction should be applied. 
A piece of apparatus may be made from a piece of stout glass tubing 
with inside diameter 18 to 20 mm. with one end drawn out to fit a 
small rubber bulb. (Bulbs sold by stationers for cleaning fountain 
pens are convenient) . This is removed when the blood or serum flows 
freely. The irritation serum which exudes after blood flow ceases is 
transferred to a clean cover-slip with a fine capillary pipette or a sterile 
platinum loop. The serum should be allowed to dry when a stain is 
employed, but when the dark-field illuminator is used, the moist pre- 
paration is diluted with a drop of normal salt solution, mounted and 
examined immediately. 

Examination with the Dark-field Illuminator.— Two types of con- 
densers are furnished by manufacturers, one of which is held on the 
microscope stage with clips, while the other is substituted for the Abbe 
condenser. Directions for setting up the illuminator are furnished by 
the manufacturers. An intense light, such as that from a small arc 
lamp, is reflected from the flat side of the mirror. The entire oil- 
immersion lens is removed from the brass collar and screwed onto a 
similar collar supplied with the condenser, fitted with a funnel stop to 
diminish the intensity of the light. The remounted lens is then placed 
on the .microscope and used for the examination. Oil is placed on the 
center of the condenser, the light adjusted so that the microscopic field 
is illuminated properly and the draw tube of the microscope pulled out 
to the proper length. Object slides should be prepared for mounting 
the serum, by making in the center circles with cedar oil about 1 cm. 
in diameter. The apparatus should be prepared before obtaining 
serum. 

Irritation serum is placed on cover-slips as directed, the cover-slips 
are turned over and mounted on the slides so that the serum is enclosed 
between the slide and cover-slip in a ring of cedar oil. A drop of cedar 
oil is put on the cover-slip and the slide then placed on the dark-field 
illuminator. When the slide has been made properly, the light rays 
pass from the condenser successively through oil, slide, irritation 
serum, cover-slip, oil, and finally the lens. 

While focussing, great care must be taken not to force the lens 
through the cover-glass. It is better to watch from the side while 
lowering the lens into the oil, then to move the lens upward with the 



398 OBTAINING IRRITATION SERUM FOR EXAMINATION 

eye at the ocular. The treponema pallidum 1 appears in a dark field as 
a white, tightly curled organism swimming actively with corkscrew 
motion. The coils, which are close and regular, number from 6 to 26. 
The length is from 10 to 20/*, and can be estimated in preparations by 
comparison with the diameter of the red blood corpuscles (average 
diameter 7/i) (Fig. 130). 

Giemsa Stain. — The irritation serum is allowed to dry in the air and 
is fixed by flooding the cover-slip with absolute methyl alcohol which 
is allowed to remain for two or three minutes when it is whisked off 
and the cover-slip dried in the air. 

The stain is prepared by mixing 10 drops of Giemsa stain, 1 drop of 
1 per cent, potassium carbonate solution and 10 cc. of distilled water. 
This is placed in a Stender dish. The cover-slip is carefully floated, 




Fig. 130. — Treponema pallidum appearing as bright refractive body on a dark fluid 
as shown by India ink or ultramicroscope. (Park and Williams.) 

film side down, on the surface of the stain. This may be done by lower- 
ing the cover-slip on a piece of fine wire bent to form a wide loop. The 
dish is covered and the cover-slip allowed to stand for twenty-four hours, 
when it is removed, washed carefully with distilled water and dried in 
the air without blotting. Mounted in balsam, it serves as a permanent 
preparation. The treponemata stain deep pink. 

1 The terminology of the organism lias been a moot question and even now there is 
no real agreement. A discussion of the nomenclature is given by Clifford Dobell. The 
organism was discovered by Schaudinn, who called it Spirochete pallida. lis specific 
name is then properly pallida, Schaudinn, 1905. The generic name Spirochete was 
not correct, however, and several others have been proposed, including Spironema, 
Treponema, and Microspironema. Since the name Spironema lias priority in time over 
the oilier-, it is held by Dobell that it must be accepted as correct unless it can lie 
shown to be unavailable. The opinion of systematists, he states, is that the spiro- 
chetes, including thai of syphilis, are nol related to the Protozoa, as the name Tre- 
ponema implies, but to the Bacteria, and that the name Treponema is not justifiable. 



EXAMINATION OF SEMEN 399 

Wright's Stain.— Wright's stain may be employed. (See page 440). 
Staining should be allowed to proceed longer than with blood, for ten 
or even fifteen minutes. 

India-ink Method.— In Burri's method a drop of irritation-serum is 
mixed with a small drop of India-ink at one end of an object-slide. 
The mixture is spread on the slide with a second slide (see page 451). 

A good grade of India-ink should be employed, since the method has 
been criticized on account of artefacts, presumably fibers of some sort, 
which resemble treponemata. The smear which should be dark- 
brown in color, is allowed to dry and is examined with an oil-immersion 
lens. The organisms appear as light spirals on a dark-ground. 

DETECTION OF ORGANISMS IN SOFT CHANCRE. 

There is no method of making a diagnosis between the so-called 
"soft chancre" of non-specific nature and the "hard chancre" of 
syphilis except by finding the Treponema pallidum. The names are 
inappropriate since many so-called "soft chancres" are found to 
harbor treponemata. Syphilis cannot be excluded simply because 
no treponemata are found on one or two occasions, for their absence 
may be due to a number of factors, such as local treatment, formation 
of scar tissue, or faulty technic. 

The bacillus of Ducrey has been found frequently in soft chancres. 
It is a Gram-negative small bacillus measuring about 0.5 by 1 to 2/x, 
occurring in chains of considerable length, and growing on blood agar. 

A safe clinical rule is, to make the diagnosis of a non-syphilitic lesion 
only after syphilis has been excluded by clinical observation supple- 
mented by repeated laboratory examinations (search for the trepone- 
mata and Wassermann reactions on the blood), extending over a period 
of at least twelve weeks. 

It has been shown recently that many of the "soft sores" are accom- 
panied by the spirochete and fusiform bacillus identical with or similar 
to those found in Vincent's angina. Whether or not these stand in the 
relation of causative factors in certain cases is not known, but their 
recognition is a matter of some importance, since suitable treatment 
often results in prompt healing. After cleaning the lesion with physio- 
logical salt solution, some of the membranous material may be removed 
with a dull curette, spread between two slides, and stained as directed 
for the organisms of Vincent's angina, or the material may be mixed 
with a drop of sterile normal salt solution and examined with the 
dark-field illuminator. 

EXAMINATION OF SEMEN. 

Spermatozoa.— Examination of semen is sometimes required in cases 
of sterility. A thermos bottle should be heated by filling it with water 
somewhat above blood heat. This is poured out, and the condom 
containing the semen placed in the bottle. The specimen should be 
examined without unnecessary delay. A drop of the material is placed 



400 EXAMINATION OF BRAIN TISSUE 

on a warm slide and covered with a cover-slip. Normally, actively 
motile spermatozoa should be seen in considerable numbers. They are 
about 45 to oO^i in length with an oval head followed by a cylindrical 
middle piece tapering to form a long tail which shows whip-like motion 
in fresh specimens. 

In the semen there are also found epithelial cells, corpora amylacea, 
and lecithin globules. The latter give semen its milky appearance. 
Under abnormal conditions, red and white blood corpuscles may be 
seen. 

Florence Reaction.— To identify stains due to semen, the Florence 
reaction may be used. The reagent consists of iodine, 2.54; potassium 
iodide, 1.65; distilled water, to 100 cc. The iodine should be washed 
with distilled water before the mixture is made. The stain is scraped 
or cut off from the material on which it is found and placed on an object 
slide. After adding a few drops of the reagent, the mixture is covered 
with a cover-slip and examined promptly under the miscroscope. 
Dark brown crystals are formed. These are lance-shaped or rhombic 
in form and may be grouped to form rosettes. The reaction is given 
not only by human and animal semen but also by various organ 
extracts, extracts from seeds, etc. It is evident therefore that while a 
negative result has some significance, a positive one has none. 

EXAMINATION OF BRAIN TISSUE FOR NEGRI BODIES. 

It has been found that Negri bodies may be found in the nervous 
system of almost all animals suffering with rabies. These are found 
more readily in the large ganglion cells of the hippocampus major. The 
bodies are regarded as of protozoal origin. Their detection is of great 
clinical importance since unfortunately it often happens that a pre- 
sumably rabid dog is killed before the development of symptoms which 
would permit the making of a diagnosis. In such cases, the dog's brain 
should be examined for Negri bodies, so that when they are present, 
the individual who has been bitten may be given Pasteur treatment 
at once. 

The animal's cranium is opened and a small bit of brain substance 
removed from (1) the convolution around the crucial sulcus; (2) the 
gray matter of the cerebellum; (3) Amnion's horn. Each particle is 
covered with a cover-glasswhichispressed down to flatten out the tissue 
and then dragged along the slide so that the cells will be separated. 
Slides may be made by the "impression method," touching the brain 
tissue to slide in a number of places so that a few cells are left in each 
area. After the tissue has dried, it is fixed for ten seconds with neutral 
methyl alcohol to which 0.1 per cent, picric acid has been added. This 
is blotted off gently and the slide covered with William's modification 
of Van Giesen's stain: 

Saturated alcoholic solution of fuchsin 5 cc. 

3 burated alcoholic solution of methylene blue 10 " 

Distilled water 30 " 



ANIMAL INOCULATION 401 

The stain keeps a long time in the refrigerator. 

The stain is heated until it steams and is washed with water, and the 
slide is examined with the oil-immersion lens. The Negri bodies are 
seen within the ganglion cells. Their size is variable, ranging from 1 
to S/jl in diameter up to Q/j, in width by 27/j. in length. The smaller 
forms are seen in animals killed with "fixed virus," and the larger ones 
in animals with " street virus." One or more nuclei are present. The 
bodies stain magenta, the red blood cells yellow or salmon color and the 
body of the nerve cell blue. Animal inoculation may be carried 
out as a confirmatory test. A salt-solution emulsion is made of 
the brain or spinal cord of the infected animal and injected through a 
trephined opening in the skull of a rabbit. Since diagnosis was 
delayed fifteen to twenty days, Wilson recommended the use of guinea- 
pigs, which develop symptoms much more quickly. 



ANIMAL INOCULATION. 

The technic of mouse inoculation for pneumococcus typing has been 
described (page 363). The technic for intravenous injection of rabbits 
has been given in the portion of the chapter on the Examination of the 
Blood which deals with immunological methods. For the detection 
of small numbers of tubercle bacilli, a guinea-pig is inoculated with 
some of the infectious material. Small young animals should be 
selected. When the material is fluid and a sediment can be obtained, 
centrifugalization should be employed in order to secure concentration, 
and the sediment should be injected. When the material is free from 
contamination and of such a nature that few tubercle bacilli are to be 
expected (as in the case of pleural fluids), about 10 cc. may be injected 
intraperitoneally. If, on the other hand, the material be purulent, 
a much smaller quantity should be injected subcutaneously in the 
inguinal region. If it be necessary to inject contaminated fluid 
intraperitoneally, as may be the case with urine, a number of animals 
should be employed. At least two pigs should be injected. 

Should the animal be living at the end of six to eight weeks, it should 
be killed. After death, the animal is tied down on a small board on its 
back with extremities drawn out with cords to the four corners of the 
board. The fur is plucked out in the median line from the sternum to 
the pubis and the skin soaked with alcohol. If cultures be made, the 
line of incision should be burned with a hot instrument and sterile 
instruments should be used, but if merely an inspection for tubercles 
be desired, these precautions are unnecessary. The peritoneum is cut 
open, the intestines are removed in toto, and search is made for gray 
tubercles or for yellowish-gray cheesy lymph nodes. Suspicious tissue 
may be removed, transferred to a slide, crushed with another slide, 
and stained for tubercle bacilli. 

26 



402 



EXAMINATION OF MILK 



EXAMINATION OF MILK. 

Human Milk.— The examination of the milk of nursing mothers is 
sometimes desirable in order to determine its suitability for infant 
feeding. At least 10 cc. of milk should be obtained with the breast 
pump. Examination may be conducted with the aid of Holt's appara- 
tus, consisting of a small hydrometer (Fig. 131, A) and a cream gauge, 
(Fig. 131, B) which is in reality a 10 cc. glass-stoppered cylinder gradu- 
ated in 0.1 cc. The reaction should be taken with litmus paper and 
the specific gravity with the hydrometer as is the specific gravity of 
urine. 




O-^lO 

l— i-9 






r a 



el D 



Fig. 131. — Glassware for milk testing. A, hydrometer; B, Holt's cylinder; C, centrifuge 
tube for human milk; D, bottle for milk testing; E, bottle for cream testing. 



For fat determinations the 10 cc. cylinder is filled with milk to the 
10 cc. mark and allowed to stand at room temperature. At the end 
of twenty-four hours, the cream will have risen to the top. The per- 
centage is read. The fat may be determined from this by multiplying 
by 0.6, since it constitutes 60 per cent, of cream. 

A modification of the Babcock method may be used for the deter- 
mination of the fat content. A special centrifuge tube is necessary 
(Fig. 131, C). This fits into the shield of the ordinary medical centri- 
fuge. Five cc. of milk are placed in the tube with a pipette, and 1 cc. 
of a mixture of equal parts of amyl alcohol and hydrochloric acid is 
added. (While this mixture may keep for several weeks, it is not 



EXAMINATION OF MILK 403 

suitable for use after it has turned dark.) Sulphuric acid (specific 
gravity, 1.83) is added cautiously to the zero mark (see directions for 
Babcock test on dairy milk) and the tube is centrifugalized for about 
three minutes at a rate of 1000 revolutions per minute or until the fat 
has risen to the top of the tube." The amount may be read on the 
scale, each division representing 0.2 per cent. 

Normally, there is 7 per cent, of cream or 4.2 per cent, of fat. The 
specific gravity is from 1.028 to 1.032. A diminution in specific 
gravity may be due either to an increase in the fat content or an 
increase in the amount of total solids. 

The protein content may be determined by placing the milk in a small 
separatory funnel in the incubator for twenty-four hours. At the end 
of this time, the cream will have risen to the top and the thin liquid 
below, containing protein and lactose, may be drawn off. After cool- 
ing, it is diluted with 9 parts of water and used for the Esbach test, 
which is carried out as directed for urine. There is about 1.5 per cent, 
of protein in normal milk, although the amount is much higher immedi- 
ately after delivery. 

For the determination of lactose, milk should be acidified with acetic 
acid, boiled, and filtered to remove the coagulated proteins. The 
filtrate is used for the determination of sugar by Benedict's method, 
preferably using Myers' technic on account of its adaptability to small 
quantities (page 245). Ten cc. of Benedict's solution are reduced by 
0.027 gm. of lactose. Normally, there is 7 per cent, of lactose in human 
milk. The amount is less immediately after the birth of the child. 

Dairy Milk. — Dairy milk is occasionally sent to the clinical laboratory 
for examination. The specimen should be thoroughly mixed to ensure 
even distribution of the cream, by pouring the milk from the bottle 
into a large beaker and back into the bottle several times. Should the 
milk come in a can, it should be poured back and forth from one can 
into another. For determination of fat content, the Babcock test is 
usually employed. This requires the use of special bottles which may 
be centrifugalized in special shields in a heavy centrifuge. Shields 
may be obtained to fit the large head of the centrifuge recommended 
for immunological work. Place 17.6 cc. of milk in the bottle with a 
special pipette (Fig. 131, D), holding the bottle in a slightly tilted 
position with the tip of the pipette into the neck, leaving an exit for 
the air in the bottle. None of the milk should be lost in the process of 
transfer. Add 17.5 cc. sulphuric acid (specific gravity between 1.82 
and 1.83) from a cylinder or pipette, again holding the bottle at an 
angle and allowing the acid to flow down the wall. The acid must be 
run into the center of the milk as a fine stream. After the procedure 
has been properly carried out, there should be two distinct layers with 
only a slight band of charred milk between them. The layers should 
be mixed by a combined rotary and shaking motion. Curds should 
not be allowed to get into the neck of the bottle. The process of 
mixing should not be interrupted until the curds which form at first 



404 EXAMINATION OF MILK 

have been completely separated. The mixture turns dark brown and 
becomes hot due to the action of the acid. The bottles are centri- 
fugalized about five minutes at a rate of 600 to 1200 revolutions per 
minute, or until all the fat has been brought to the surface of the liquid. 
It may be necessary to fill the centrifuge shields with hot water to keep 
the fat liquid. "When separation is complete, hot water is added with 
a pipette to bring the upper level of the fluid to a point near the gradua- 
tions on the neck. The bottle is centrifugalized for one minute, hot 
water added again so that the lower level of the fat will come within 
the graduated scale, and the bottle centrifugalized for a minute. The 
amount of fat may be read on the scale. It is convenient to use a 
pair of dividers, measuring from the top of the fat column to lower line 
of separation between fat and water. It is important to note that the 
reading is taken at the top of the meniscus. The dividers may then 
be moved to a convenient place on the scale and the amount of fat read. 
Each division of the scale represents 0.2 per cent. 

Different types of bottles should be used for testing skimmed milk 
and cream. The bottle suitable for skimmed milk has a neck with 
smaller internal diameter and the divisions of the scale represent one- 
twentieth of 1 per cent. The bottles for cream have a much wider 
neck and the scales read up to 30 per cent. (Fig. 131, E). In testing 
cream, 18 cc. instead of 17.6 cc. should be used, on account of the lower 
specific gravity. 

The specific gravity of the acid may be determined by weight. A 
test bottle is dried, weighed, filled to the zero mark with sulphuric acid 
and weighed again. The difference between the two weights repre- 
sents the weight of acid. The acid is poured out and the bottle rinsed 
with several changes of water until the water fails to redden blue 
litmus. Distilled water is poured in to the zero mark and the bottle 
weighed again. From this weight is subtracted the weight of the 
empty bottle to obtain the weight of water. The specific gravity of 
acid is obtained by dividing the weight of the acid by the weight of the 
water. The quotient should fall between 1.82 and 1.83. Care should 
be taken to dry the outside of the bottle each time. 

Tests for Preservatives.— Formaldehyde.— The tests for formaldehyde 
are really general tests for the aldehyde group: (1) Three cc. of milk 
are diluted with an equal quantity of water, and the mixture is placed 
in a test-tube. Sulphuric acid (specific gravity, 1.82-1.84) is allowed to 
run from a pipette to form a layer under the milk, just as nitric acid is 
run in when performing the nitric acid test for albumin in urine. If 
formaldehyde be present, a violet ring is formed at the junction of the 
two liquids. This color reaction may be noticed in carrying out the 
Babcock test. (2) Five cc. of milk are placed in a white porcelain 
evaporating dish with an equal quantity of water. To this are added 
10 cc. of hydrochloric acid and a few drops of 10 per cent, ferric chloride. 
A violet color is seen in the presence of formaldehyde. 



CARBON-DIOXIDE TENSION OF THE ALVEOLAR AIR 405 

Boric Acid.— Ten cc. of milk are alkalinized with 10 per cent, sodium 
hydrate and evaporated. The residue is incinerated and the ash 
moistened with enough very dilute hydrochloric acid to give it a dis- 
tinctly acid reaction. A piece of turmeric paper is moistened with the 
solution. The paper is then dried on a watch-glass placed over a boil- 
ing water-bath. When boracic acid or borates are present, the paper 
turns reddish-brown on drying. 

Bacteriologic Examination of Milk.— The specimen should be thor- 
oughly mixed as directed for the Babcock test, using vessels which have 
been carefully cleaned. The milk is diluted by adding 1 cc. to 100 cc. 
of sterile bouillon. Sterile pipettes should be used for measuring these 
quantities. Each cc. of the dilution contains 0.01 cc. of milk. 

Culture tubes containing nutrient agar should be placed in a water- 
bath with boiling water to liquefy the media. The temperature of the 
water in the bath should be reduced to 43° C. Some of the milk dilu- 
tion is added to each tube, 1 cc. to one tube (A), 0.1 cc. to a second 
(B), and 0.01 cc. to a third tube (C). When there is reason to believe 
that the milk is unusually dirty, 1 cc. of the 1 : 100 dilution may be 
added to another 99 cc. of sterile bouillon. This will give a 1 : 10,000 
dilution, 0.1 cc. of which may be added to a fourth tube ( D) The 
medium and the milk in each tube are thoroughly mixed by rolling the 
inoculated tubes between the palms of the hands, keeping the tubes 
upright. The mouths are then passed through the flame and the 
contents of each tube poured into a Petri dish. The plates are covered 
and, after the medium has solidified, are inverted and placed in the 
incubator at 37° C. At the end of forty-eight hours, the number of 
colonies is counted. A plate should be selected for counting which 
contains less than 100 colonies. A ruled plate counter is convenient. 
In estimating the number of bacteria, the number of colonies is multi- 
plied by the dilution. The following factors may be employed when the 
dilutions are made according to the method which has been given: 
Tube A, 100; Tube B, 1000; Tube C, 10,000; Tube D, 100,000. For 
example, 20 colonies on the plate made from Tube B means that there 
are 20,000 bacteria in 1 cc. of milk. 



CARBON -DIOXIDE TENSION OF THE ALVEOLAR AIR. 

The significance of reduction in the carbon-dioxide tension of the 
alveolar air has been referred to in the discussion of tests for acidosis 
in the blood (see pages 198 and 211). The tension is dependent upon 
the C0 2 content of the blood and the findings follow those which are 
obtained in determining the C0 2 combining power of the blood. 

Fridericia Method.— Apparatus and Reagents Required. 1 —^) Frideri- 
cia apparatus (Fig. 132). There should be two stop-cocks, the upper 
one an ordinary two-way cock and the lower one a three-way cock of 

1 An accessible description is given by Poulton. 



406 CAEBOX-DIOXIDE TENSION OF THE ALVEOLAR AIR 




wide bore (10 mm.). The volume should be 100 cc. If not exactly 

100 cc, then the scale should be graduated in percentages of the actual 
volume up to 8 per cent.; (2) rubber bulb to fit outlet; (3) water-bath 
or pail in which the Fridericia apparatus may be stood upright; (4) 
sodium hydrate, 20 per cent, solution; (5) dilute acetic acid. 

Principle.— The alveolar air is exhaled and 100 cc. is caught and 

retained in the portion of the apparatus between stop-cocks A and B. 

Sodium hydrate is drawn in to absorb C0 2 . The amount of the gas 

absorbed is measured by the difference 

in volume of the alveolar air, which 

may be read on the scale. 

Procedure. It will be convenient to 
describe the three positions in which 
stop-cock B may be placed. 

Position I. X and Y communicate, 
the outlet C being closed. 

Position II. Y and C communicate, 
and X is cut off. 

Position III. X and C communicate 
while Y is cut off. 

The patient is instructed to breathe 
into the apparatus through the mouth- 
piece of arm X, the cock A being open 
and B being in position I, so that X and 
Y communicate while C is shut off. At 
the end of a normal inspiration and 
without unusual pause, he is directed to 
blow one hard breath quickly through 
the apparatus. Cock A is closed while 
B is left unchanged. By this process 
all the atmospheric air is displaced from 
the apparatus, which is now filled with 
alveolar air. The apparatus is placed 
in an upright position in the pail of 
water at room temperature, leaving the 
openings of both Y and X out of the 
water. On cooling, some of the alveolar 
air from Y is drawn into X but there is 
no clanger of the atmospheric air drawn 
into the upper part of Y reaching X. In five minutes the stop-cock C 
is turned to Position II, so that X is shut off but Y communicates 
with C. A rubber bulb is attached to the top of Y and about 2 or 
3 cc. of the alkali are drawn into the arm Y through the opening C. 
The cock B is now turned back to Position I so that X and Y com- 
municate. The soda is forced into X with slight pressure from the 
bulb. A, of course, should be closed during this procedure. While 
performing this step, the arm Y is depressed a little and the arm X 



B 



Fig. 132. — Fridericia apparatus. 



CARBON-DlOXibE TENSiON OF THE ALVEOLAR AIR 407 

tipped up so that none of the alveolar air can escape. After this the 
cock B is turned back to Position II so that the soda in arm Y may run 
out through the outlet C and so that X will be closed completely. 
The apparatus is inverted and shaken gently for a half-minute or so 
to expedite the absorption of the C0 2 by the soda, when it is returned 
to the water-bath and the stopcock B is turned under water so that X 
and C communicate (Position III), so that the water in the bath may 
run into the arm X. The reading is taken at the end of five minutes. 
The apparatus is raised so that the water in X and that in the bath are 
at the same level and the level of the water meniscus in X is read off 
on the scale. 

No correction need be made for absorbed water vapor since this is 
small and falls within the limits of error of the method. To transpose 
the reading to millimeters of mercury under corresponding conditions 
of temperature and water vapor, it is only necessary to multiply the 
percentage found by the barometric pressure expressed in millimeters 
of mercury. When a reading has been made, the apparatus should be 
cleaned by rinsing with a little dilute acetic acid. 

Normal Reading. — The normal reading is a percentage of 5.5, which 
at sea level corresponds to 42 mm. of mercury. 

Marriott's Method.— This has the advantage of being more convenient 
for bedside determinations, and provides a means for the collection of 
the specimen from infants and comatose adults. 

Principle. When the air, charged with C0 2 , is passed through a 
solution of sodium carbonate until the solution is saturated, the solu- 
tion contains sodium bicarbonate and dissolved C0 2 , the final reaction 
depending upon the relative amounts of sodium bicarbonate and acid 
C0 2 . This depends entirely upon the tension of the C0 2 in the air 
which is blown in, and when saturation has been attained is quite 
independent of the amount of air. The reaction of the resulting solu- 
tion is changed to the acid side by high tensions of C0 2 , to the alkaline 
side by low tensions, so that the reaction may be used as a measure of 
the C0 2 tension. In the test, phenolsulphonephthalein is used as the 
indicator and the standards for comparison are made by mixing definite 
proportions of acid and alkaline phosphate solutions. 

Apparatus and Reagents Required.— (1) Rubber bag of approximately 
1500 cc. capacity connected by a short rubber tube to a glass mouth- 
piece, and a pinch-cock; (2) atomizer bulb; (3) glass tube or pipette 
drawn out to a fine point; (4) box for color comparison, similar to that 
used in the Sahli hemoglobinometer but provided with three holes 
instead of two, with a background of ground glass or opal glass; (5) 
eight test-tubes containing standard phosphate solutions; (6) standard 
bicarbonate solution; (7) test-tube of same diameter as the tubes con- 
taining the standard solutions, but 100 to 150 mm. long; (8) mask for 
infants or for comatose patients. This may be adapted from the mask 
used for ether anesthesia, or may be improvised according to the 
suggestion of Howland and Marriott. They suggest the use of the 



408 CARBON-DIOXIDE TENSION OF THE ALVEOLAR AIR 

rubber mouth-piece of a Hygeia nursing bottle. A piece (about 8 by 
10 inches) of thin rubber tissue such as that used by dentists for a dam 
is perforated in the center with a piece of hot metal. The hole which 
is made in this manner is stretched and pulled down over the nipple 
as near the edge as possible and is cemented with rubber cement, 
reinforcing the union by applying a strip of adhesive plaster about 
three-eighths or half an inch wide around the edge of the nipple to 
overlap the rubber tissue. The tip of the nipple is cut off and a short 
glass tube, about three-eights inch in diameter, is tied into the resulting 
hole. 

Preparation of Standard Phosphate Solutions.— Two solutions are 
necessary, one a fifteenth-molecular solution of acid potassium phos- 
phate and the other a fifteenth-molecular solution of alkaline sodium 
phosphate. These are made in the same manner and in the same pro- 
portions as the solutions for determining the alkali reserve of the blood. 
(See page 208.) To them, phenolsulphonephthalein is added as an 
indicator as there directed. The solutions are mixed in the propor- 
tions which are indicated in the table below. The horizontal column 
bearing the legend "mm." gives the value of the particular solution 
in terms of carbon dioxide tension when expressed in millimeters of 
mercury (see Fridericia method). 

PROPORTIONS IN WHICH PHOSPHATE SOLUTIONS ARE MIXED. 

Mm. 10 15 20 25 30 35 40 45 

Acid potassium phosphate, cc. . 17.8 25.2 31.0 35.7 40.5 45.0 47.0 50.2 
Alkaline sodium phosphate, cc. . 82.2 74.8 69.0 64.3 59.5 55.0 53.0 49.8 

The solutions may be kept unaltered for some time. They should be 
preserved, however, in glass which does not give off alkali readily, 
such as Pyrex, Non-sol, or Jena. Comparison tubes should be of the 
same sort of glass. To prevent the growth of moulds, a little thymol 
or tuluol may be added to each solution. 

When the solutions have been mixed, a small quantity of each should 
be placed in a small tube, about 10 by 75 mm. The tubes should be 
properly labelled, sealed, and kept in a dark place. 

The standard bicarbonate solution may be made by dissolving 0.53 
gm. of desiccated sodium carbonate in a little water. To this is added 
200 cc. of 0.01 per cent, aqueous solution of phenolsulphonephthalein 
and then enough distilled water to bring the total volume to one liter. 
If desired, 100 cc. of decinormal sodium hydrate may be placed in a 
liter flask, 200 cc. of 0.01 per cent, phenolsulphonephthalein added and 
then water to bring the total volume to the liter mark. This latter 
solution may be used as it is, since when the carbon dioxide from the air to 
be tested is blown through, the alkali will be converted to carbonate. 

Collection of Alveolar Air.— With an adult who is capable of cooperat- 
ing to some extent, Marriott recommends the method of Plesch, 
modified by Higgins. About 600 cc. of air are blown into the rubber 
bag with the atomizer bulb. The rubber tube is then closed with the 



CARBON-DIOXIDE TENSION OF THE ALVEOLAR AIR 409 

clip. The patient, who should be at rest, is directecTto breathe easily 
and naturally and to avoid taking a deep inspiration just before the 
collection of the specimen is started. At the end of a normal expiration 
the mouth-piece is placed in his mouth and his nostrils are closed by 
the operator. He is allowed to breathe back and forth into the bag 
four times, drawing the air from the bag into his lungs with each 
inspiration. The operator should indicate the time for inspiration and 
expiration. At the end of twenty seconds, the tube leading from 
the mouth-piece to the bag is clamped. The analysis should be made 
within three minutes since there is a considerable loss of carbon 
dioxide through the rubber. 

When alveolar air is to be collected from children or from uncon- 
scious adults, the special mask should be employed. This should be 
attached to the bag with the tubing. With comatose adults, the bag 
should be filled with about 1000 cc. of air and the period of breathing- 
lengthened to thirty seconds, since the bag cannot be emptied with 
each inspiration. 

With infants under one year, the amount of air in the bag should be 
from 250 to 400 cc, enough so that the bag is never emptied com- 
pletely with the inspiratory effort. The infant should have been quiet 
for at least one minute previous to beginning the collection of the 
sample, since vigorous crying leads to a lowering of the tension. While 
the infant usually cries during the collection of the specimen, this serves 
to facilitate the mixing of the gases. 

After the bag has been filled with the proper amount of air and has 
been closed with the pinch-cock, the mask is placed over the face, as 
nearly as possible at the close of expiration. The rubber tissue should 
be fitted closely about the face to prevent the loss of air, the clamp 
is opened and respirations are permitted to go on from twenty-eight to 
thirty-two seconds, when the tube is clipped and the mask removed. 

Method of Making Determination.— Into a test-tube of the same 
diameter as those containing the standard solution but about 100 to 
150 mm. long is placed about 3 cc. of the standard carbonate or sodium 
hydrate solution, containing phenolsulphonephthalein. The piece of 
glass tubing which has been drawn to a fine point is inserted into the 
rubber tube attached to the bag. The capillary end of this piece of 
tubing is then placed in the solution and the clip is released so that 
the air may bubble through the carbonate solution until there is no 
further color change. The tube is then stoppered and compared 
immediately with the standard tubes. If the tube does not match 
any of the standard tubes exactly, it is placed between the tubes which 
it most nearly matches and the result is read by interpolation. The 
readings are in terms of millimeters of mercury. The solutions are 
prepared to give readings at a temperature between 20 and 25° C. 
When the room temperature is much higher or lower, the tubes should 
be placed in water at 25° C. while the air is being bubbled through, 
when they are removed for color matching. 



410 CARBON-DIOXIDE TEXSIOX OF THE ALVEOLAR Ml: 

Findings. — The C0 2 tension in normal adults at rest ranges from 40 
to 45 mm. According to Marriott, tensions between 30 and 35 mm. 
are indicative of a mild degree of acidosis, while a tension below 20 mm. 
is regarded as a sign of imminent danger. The tension of infants runs 
from 3 to 5 mm. lower than that of adults. 

The C0 2 tension may be affected by conditions other than acidosis. 
Whenever the respiratory center is stimulated, as by caffein, there is 
increased pulmonary ventilation and the tension is lowered; while when 
the center is depressed, the tension is increased. 



CHAPTER VII. 
BACTERIOLOGICAL METHODS. 

General Considerations.— Bacteriological examination is undertaken 
to determine whether or not microorganisms be present, their nature, 
and number. The methods include the microscopic study of material 
obtained from the body (exudates, etc.), the use of cultures, and animal 
inoculation. The technic for animal inoculation has been given 
elsewhere. 

Direct Microscopic Examination.— In some instances, microscopic 
examination alone serves clinical purposes (e. g., detection of tubercle 
bacilli in sputum) . The material is spread upon a slide to form a thin 
film, which is dried, fixed, and stained. Suitable stains have been 
indicated in the foregoing portions of the text describing the examina- 
tion of various exudates. General staining methods will be summarized 
in a following paragraph. 

A "hanging drop" preparation may be made for determination of 
motility. A slide with a circular concavity is usually employed. 
The surface of the slide around the hollow is smeared with a little vase- 
line or cedar oil. A drop of the culture is placed in the center of a 
cover-slip with a platinum loop. The cover-slip is turned over quickly 
so that the drop hangs from the lower surface, and placed over the 
hollow in the slide. When properly mounted, the drop hangs in a 
sealed chamber. If preferred, the drop may be mounted on a slide, 
which is inverted in a moist chamber. 

Cultural Methods.— Collection of Material.— Care must be taken to 
obtain the material free from outside contamination. It should be 
secured in a sterile receptacle and transferred to appropriate culture 
media with sterile instruments. Pus is usually secured on sterile cotton 
swabs. The swab is replaced in the sterile tube which serves as a 
container. For inoculation, the swab is wiped over the surface of solid 
medium or dipped into liquid medium. 

When few bacteria are present, as in chest fluid, larger quantities of 
material should be obtained in sterile vessels (bottle, flask, or test- 
tube), and transferred to appropriate media with a pipette. The 
technic for blood-cultures has been described elsewhere. Cultures are 
valueless unless the material has been collected in sterile containers. 

Inoculation of M ed ia.— Inoculation may be made into flasks or tubes 
of liquid media, or into tubes or plates of solid media. When few 
bacteria are present in thin, fluid material, a considerable quantity 
should be transferred to the media with a pipette. Pus may be 



11! 



BACTERIOLOGICAL METHODS 



inoculated into liquid media with the cotton swab or platinum loop 
(Fig. 133, b). The inoculating loop must be heated and eooled before 
and after it comes in contact with infectious material. A sterile cotton 
swab or platinum spreader is more convenient for making smears on 
plates, since it is less likely to cut the surface of the medium. 

Incubation.— Inoculated media should be placed in an incubator the 
temperature of which is maintained at constant point by heat-regulat- 
ing mechanism. The optimum temperature for most pathogenic 
organisms is body temperature (37.5° C.). Gelatin, however, must be 
kept at lower temperature, usually that of the room. ^Yhen electricity 
is available, an electrically heated and regulated incubator is preferable 




Fig. 133. — Bacteriological apparatus, o, swab tube; b, inoculating loop; c, inoculating 
needle; d, fermentation tube; e, Durbam fermentation tube; /, arrangement for tubing 
medium. 



to gas on account of the freedom from fire hazard and the constancy of 
temperature. 

Study of Cultures.— The cultures are examined after incubation for 
suitable length of time, which varies with different organisms, but is 
ordinarily from twenty-four to forty-eight hours. The following 
methods are at our disposal: (1) Examination of cultures by naked 
eye; (2) examination of individual colonies by naked eye and with 
hand lens; (3) Chemical changes in culture medium; (4) morphology 
and staining reactions of organisms; (5) motility; (6) requirement of 
anaerobic conditions for growth. 

In practical work it is well to examine the culture microscopically 



CULTURAL METHODS 413 

first to determine if it be pure or mixed. If pure, the study of the 
cultures may be completed. If morphological examination of a stained 
film, however, shows more than one form of organism, the varieties 
must be isolated. 

In the study of cultures, the gross appearance of liquid media should 
be noted, including turbidity, pellicle, sediment, gas formation, and 
odor. Colonies on solid media should be described in detail, observing 
the size, conformation of borders, color, transparency, texture, eleva- 
tion, and moisture. 

Microscopic examination is important and should include at least a 
study of films stained with Loeffler's methylene blue solution and with 
Gram's stain. Special methods should be employed for capsules, polar 
and other bodies, spores, and flagella when indicated. Motility should 
be determined by the examination of a "hanging drop." Among the 
chemical changes which may be seen and which are helpful in differ- 
entiation are, coagulation of milk, change in reaction of media, forma- 
tion of gas and change in reaction in sugar media by breaking down of 
sugar, and production of indol. 

Isolation of Pure Cultures. — For isolation of varieties, solid media 
must be employed, so that separate colonies are obtained, from a 
number of which inoculations may be made into media to obtain pure 
cultures. The purpose is to secure dilution and distribution of material 
on solid media so that the colonies will be discrete. Streak plates may 
be made by drawing the inoculating wire or platinum spreader over the 
surface of a number of tubes of solid media, or in a series of streaks over 
one or more plates. In this way the material is thinned. A second 
method employs "poured" plates. Agar tubes are melted by placing 
them in boiling water. The temperature of the water is reduced to 
about 40° C. when the first tube of the series is inoculated with a loopful 
of the infectious material. The contents of the inoculated tube are 
then thoroughly mixed, and two or three loopsful are placed in the 
second tube. After mixing thoroughly, two or three loopsful from the 
second are carried into the third tube and so on. The mouths of the 
tubes are then passed through the flame in turn and the contents 
poured into sterile Petri dishes, lifting the covers just enough to permit 
pouring the contents of the tube into the dish. After the medium has 
solidified, the plates are inverted and placed in the incubator. A third 
method is to take a series of agar tubes and inoculate a loopful of infec- 
tious material into the water of condensation of the first tube. A loop- 
ful of this is then transferred to the water of condensation of the second 
tube. After mixing, a loopful is carried to the third tube and placed 
in the water of condensation. Then all the tubes are tipped so that the 
water of condensation runs over the surface of the agar and are placed 
in the incubator. For isolation, the colonies which grow on plates 
should be viewed with the lowest power of the microscope, placing the 
Petri dish with cover removed on the stage. Colonies are removed 
("fished") with a sterilized platinum needle (Fig. 133, c) (not loop), 



414 BACTERIOLOGICAL METHODS 

keeping the needle point and the colony in the focus of the lens, so that 
the point may be seen to touch the colony. The needle is withdrawn 
without again touching the colony or other object and the material on 
it is placed in a tube of medium. 

Staining Methods. — A thin film is made on a slide or cover-slip. Pus 
may be spread by the "impression method," by smearing the cotton 
swab over the surface of the slide, or by putting a drop of pus at one 
end of a slide and spreading it with a second slide (see method for 
making blood films with slides, page 45). In studying cultures in 
nutrient bouillon or other liquid media, a drop is placed on a slide or 
coverslip with a platinum loop (heated in the flame to incandescence 
and cooled before and after use). When solid media are employed, a 
drop of normal saline should be put on the slide and a small particle of 
the growth, picked up with an inoculating needle mixed with it. The 
slide is dried in the air, or by holding it high over (not in) the flame. 
The danger of overheating will be obviated if the slide be held in the 
fingers during this step. Fixation is accomplished by passing the slide 
through the flame a number of times with the film side uppermost. 
The film should not be allowed to scorch. When a brown color is seen, 
the preparation should be discarded. Instead of heat, the slide may be 
fixed by covering it with absolute methyl alcohol for two minutes. 
After fixing, the stain is poured on and allowed to remain the necessary 
length of time, usually one-half to one minute. Except in staining 
with carbol-fuchsin for the tubercle bacillus, heating is generally 
unnecessary. The stain is poured off, the slide washed with water, 
and dried either by blotting with blotting paper, or preferably by 
shaking the water off with a motion similar to that employed in shaking 
down a clinical thermometer, evaporating the residue with moderate 
heat. When a cover-slip has been employed, it is mounted by placing 
it over a drop cf Canada balsam on a slide, film side down. The cover- 
slip should be pressed down gently in order to spread out the balsam 
in a thin layer. When slides are employed, a cover-glass is not 
necessary. 

PREPARATION OF MEDIA. 

Tubed media may be secured through retail drug houses, though 
the price is prohibitive when much work is done. A number of manu- 
facturers offer media in dry powdered form which may be prepared 
for use by adding water and sterilizing. This is a convenient method 
for those who do not wish to carry out all steps of preparation and the 
cost is low. The greatest degree of satisfaction, however, is obtained 
by the workers when he has media prepared, titrated, and sterilized 
under his own directions. 

After the ingredients have been mixed according to formula, the 
reaction must be adjusted and the medium cleared, filtered, sterilized 
and tubed. Directions for these steps are given, followed by formula?. 



PREPARATION OF MEDIA 415 

Preparation of Glassware.— New glassware may be immersed in a 
dilute solution of nitric acid, rinsed in tap-water, then in distilled 
water, and allowed to dry. Glassware containing culture medium or 
infectious material should be boiled in a 1 or 2 per cent, solution of 
sodium carbonate and brushed with a test-tube brush, protecting the 
hands from the strong alkali with rubber gloves. The glassware is 
then rinsed with tap-water, next with very dilute acid, and finally with 
distilled water, and allowed to dry. If foreign substances cannot be 
removed by this method, which is usually sufficient, the glassware may 
be immersed in bichromate cleaning fluid : 



sium bichromate 100 gm. 

Sulphuric acid, concentrated 120 cc. 

Water 1000 " 

This solution may be used over and over again. 

Flasks, bottles, and tubes should be stoppered with raw cotton 
rolled to form plugs which will fit the mouths and which will project 
far enough to be grasped readily by the fingers. (Fig. 133, e.) 

Types of Apparatus.— Media may be tubed in ordinary test-tubes, 
with or without lip. Tubes of good glass are desirable, in order to 
prevent the liberation of alkali by repeated heating. Large square 
bottles may be used for making dilutions and as substitutes for more 
expensive flasks. Blake bottles are convenient, since when laid on the 
side, a large surface of broth or agar is afforded. Petri dishes are round,, 
flat, glass plates provided with loosely fitting glass or porous pottery 
covers. The lower portions serve to hold solidified media. The 
ordinary size is 10 cm. in diameter. They are used for the isolation of 
colonies. Ordinary milk bottles are convenient for storing media. 
Fermentation tubes with bulb and closed arm may be secured with or 
without graduation (Fig. 133, d). They are expensive and somewhat 
inconvenient to handle. For most purposes, Durham jermentation 
tubes may be substituted (Fig. 133, e). A small test-tube, about 1 by 

6 cm. is dropped, open end down, into a culture tube containing 5 to 

7 cc. of medium. The fluid completely fills the small tube during 
sterilization. After the medium is inoculated, any gas formed will rise 
in the small tube, displacing from it part of the medium. 

Swab-tubes may be made by taking pieces of copper wire or wooden 
applicators the length of which exceeds that of the ordinary test-tube, 
wrapping one end tightly with absorbent cotton, and placing the swab 
in a test-tube. The cotton closing the mouth of the test-tube is 
wrapped about the upper end of the wire (Fig. 133, a). The outfit 
may be sterilized by dry heat. 

Adjustment of Reaction.— The reaction of media is of great import- 
ance since bacterial growth is susceptible to variations in acidity or 
alkalinity. Two methods for adjusting the reaction are given here, 
the titrimetric method which has been employed for many years, and 
colorimetric determination of the hydrogen-ion concentration. 



416 BACTERIOLOGICAL METHODS 

Titrimetric Method.— With a volumetric pipette, put 5 cc. of the 
medium in a white porcelain evaporating dish and add 45 cc. of distilled 
water. Boil briskly for a minute. Add 1 cc. of phenolphthalein solu- 
tion (0.5 gm. dissolved in 100 cc. of 50 per cent, alcohol) and titrate at 
once by adding ^tt sodium hydrate solution from a burette (graduated 
in 0.1 cc.) until a faint but distinct color marks the end-point, stirring 
the medium constantly with a glass rod while adding the alkali. 
According to the standard methods of the American Public Health 
Association this tint may be described as a combination of 25 per cent, 
red (wave length approximately 658) with 75 per cent, white as shown 
by the disks of the standard color top made by the Milton Bradley 
Educational Company of Springfield Massachusetts. 

Should the medium be alkaline, that is, give a red color on the 
addition of the phenolphthalein indicator, titration should be carried 
out by adding ^ T hydrochloric acid until the pink color disappears 
and the medium becomes colorless. 

The amount of acid or alkali required for neutralization is readily 
calculated. While ^ alkali or acid solution is used for titration, the 
medium is neutralized with normal solution. It is evident that if X 
quantity of j; T XaOH be required to neutralize 5 cc. of medium, then 
X cc. normal NaOH will neutralize 100 cc, and 10 times this amount 
will neutralize 1 liter. 

Reaction is expressed with reference to the phenolphthalein end- 
point (Fuller scale). Alkaline media are recorded with the minus sign 
( — ) before the percentage of normal acid solution which would be 
required for neutralization, while acid media are recorded with the plus 
sign ( + ) before the amount of normal alkali which would be required 
for neutralization. The actual adjustment of reaction is determined 
by the use to which the medium is put. For examination of water, 
the standard methods of the A. P. H. A. specify that the reaction shall 
fall between +0.5 and +1.0. When titration shows that the reaction 
is within this range, no adjustment is necessary. For general routine 
work, slight alkalinity, neutrality or an acidity not exceeding +1.0 is 
customary. 

Example.— Suppose the desired reaction be +1.0. Titration shows 
that 2.5 jj[ XaOH is require to neutralize 5 cc. Then 2.5 cc. of normal 
XaOH is required to neutralize 100 cc. Therefore, the reaction of the 
medium before adjustment is + 2.5 (since this is the percentage of 
normal alkali required for neutralization) . A reaction of + 1 .0 is 
desired, however, so that to each 100 cc. of media, 1.5 cc. (2.5 — 1.0 = 1.5) 
of normal XaOH should be added 

Colorimetric Determination of Hydrogen-ion Concentration.— The titri- 
metric method is open to a number of objections. One is that the 
end-point is not sharp due to the action of buffer-substances (proteins, 
amino-acids, and certain salts) which take up the hydrogen ions so 
that the addition of moderate quantities of acids make a comparatively 
small change in the reaction. Another is that a great difference is 



PREPARATION OF MEDIA 417 

made by personal equation of the operator on account of variations in 
sharpness of color-sense. The true reaction depends upon the amount 
of hydrogen or hydroxyl ions which are present. Hydrogen : ion con- 
centration is best determined by the hydrogen electrode, but such appa- 
ratus is not available except in well equipped laboratories. In clinical 
laboratory practice, sufficiently accurate results can be obtained by 
colorimetric methods. (Brief consideration has been given to hydro- 
gen-ion concentration in the chapter on Blood, page 208). 

The hydrogen-ion concentration is expressed in terms of a negative 
logarithm as the "hydrogen exponent," and is usually written pH. 
With such minus logarithms, two facts should be remembered. The 
actual amount of hydrogen ions decreases as the pH increases. In 
other words, pH=8.0 represents a less acid solution than pH=7.0. 
Furthermore, the amounts are logarithms, and not simple quantities, 
so that pH = 3.0 is a solution containing 10 times as many hydrogen ions 
aspH=4.0. 

The hydrogen exponent of water is pH = 7.0, while that of blood 
fluids is in the region of pH = 7.5. It is usually desired to prepare 
media with reaction in the neighborhood of pH = 7.0 to pH = 7.5. 
While various indicators are required at different ranges, for example, 
thymol blue between pH = 1.2 and pH = 2.8, and bromphenol blue 
between pH = 2.8 and pH = 4.6, phenolsulphonephthalein (phenol red) 
is the indicator whose color changes are best appreciated within the 
range from pH = 7.2 and pH = 8.0. The initial reaction of a medium 
may be determined by preparing solutions of known hydrogen-ion con- 
centration, adding to them small amounts of the indicator. The tint 
produced serves as a standard to which the medium is brought by 
titration. A precisely similar quantity of indicator is added to a 
measured quantity of the medium to be tested and acid or alkali is 
run in from a burette until the colors of the solution and of the standard 
match. In matching the colors, the brown tint of the medium is com- 
pensated for by interposing tubes of untreated medium between the 
standards and the light. 

Procedure for Determination of Hydrogen-ion Concentration.— A 
colorimetric method was utilized by Hurwitz, Meyer, and Ostenberg. 
This is given here, except that titration is performed with 5 cc. samples 
rather than 3 cc. samples. The standard colors may be prepared accord- 
ing to the directions of Levy, Rowntree and Marriott after Sorensen. 
Two standard phosphate mixtures are necessary. T V molecular acid 
or primary potassium phosphate; 9.078 gm. of pure recrystallized salt 
(KH 2 P0 4 ) is dissolved in freahly distilled water (freed from ammonia 
by boiling) and made up to 1000 cc. 

Y 1 ^ molecular alkaline or secondary sodium phosphate. — The pure 
recrystallized salt (Na 2 P04.2H 2 0) is exposed to the air for about two 
weeks, protected from the dust. Ten molecules of water are given off so 
that the formula of the salt obtained is NaHP0 4 .2H 2 0. Of this, 
11.876 gm. are dissolved in distilled water and the volume is made up 
27 



418 



BACTERIOLOGICAL METHODS 



to one liter with water. If the salt be sufficiently pure, the solution 
should give a deep red with phenolphthalein. 

The solutions are mixed in the proportions shown in the following 
table to obtain the indicated pH: 



pH 6.4 


6.6 


6.8 7.0 7.1 


7.2 7.3 


7.4 


7.5 


7.6 


7.7 


7.8 


8.0 


8.2 


8.4 


Primary potassium 
phosphate sol., cc. ; 73 

Secondary sodium | 
phosphate sol., cc. 27 


63 
37 


51 37 32 
49 63 68 


27 23 
73 77 


19 

81 


15.8 
84.2 


13.2 
86.8 


11.0 
89.0 


8.8 
91.2 


5.6 
94.4 


3.2 
96.8 


2.0 
98.0 



Five cc. of each solution are placed in Pyrex glass test-tubes of 
similar size, 0.5 cc. of 0.01 per cent, aqueous solution of phenolsul- 
phonephthalein 1 are added to each tube, and the ends of the tubes 
sealed. 

Color comparison is made in a comparator, or wooden box with a 
double row of holes, three in a row, so arranged that the tubes are 
viewed with transmitted light. The comparator shown (Fig. 134) is a 
modification by Cole and Onslow of one suggested by Hurwitz, Meyer, 
and Ostenberg, and by Walpole. 




Fig. 134. — Cole and Onslow's color comparator. 

In addition, the following apparatus and reagents are required: 
(1) Microburette, improvised from a calibrated 1.0 or 2.0 cc. pipette, 
divided into 0.01 cc. A fine glass capillary tip should be attached to 
the lower end with rubber tubing and the delivery of liquid should be 
controlled with a pinch-cock; (2) bacteriological pipette to deliver 0.5 
cc. and a volumetric pipette to deliver 5.0 cc; (3) clean Pyrex glass 
test-tubes of same size as those used for standards; (4) phenol-red 



1 This may be prepared by adding 59 cc. of distilled water to 1 cc ( = 6 mg.) of the 
standardized solution prepared by Hynson, Westcott and Dunning, of Baltimore, Md., 
for testing kidney function. This solution should be used also as the indicator in 
carrying out the titration. 



PREPARATION OF MEDIA 419 

solution, 0.01 per cent. This should be the same solution as that used 
in making standards. (5) yr NaOH, made from the normal solution 
which is finally added to the media for adjustment of reaction. To 
prepare, place 50 cc. of -?- solution in a 1000 cc. volumetric flask and 
add distilled water to the 1000 cc. mark. Both alkali solutions should 
be kept in bottle whose inner surfaces have been coated with paraffin. 
Titration.— Measure 5 cc. of the medium into one of the test-tubes 
and add 0.5 cc. of 0.01 per cent, phenol-red solution. Place this in 
the middle hole (No. 2) of the front row in the comparator (Fig. 134). 
In the hole directly in back (No. 5) place a tube with distilled water. 
If a reaction of pH =7.5 be desired, place in the hole on the left (No. 1) 
standard tube 7.4- and in the hole on the right. (No. 3) standard tube 
7.6. In the two holes back of these (Nos. 4 and 6), place tubes con- 
taining uncolored "medium. If the medium be more acid than is 
desired, add f NaOH from the microburette until the tint is "between 
that of the two standard tubes, viewing the tubes with transmitted 
light. Shake the tube to mix contents thoroughly. When the 
smount of alkali required to bring 5 cc. of the medium to the desired 
tint is known, it is easy to calculate the amount of rf- NaOH required 
to bring the bulk of the medium to the required hydrogen-ion concen- 
tration. If, for example, 0.3 cc. of ^ NaOH were required to bring 
the desired color with 5 cc, 3.0 cc. of normal NaOH would be needed 
for 1 liter. 1 

Media become more acid after sterilization. This is particularly 
marked in media containing sugar. It is suggested that sugar solu- 
tions be sterilized separately and added to the medium after the 
nitrogenous part of the medium. Solid media may be liquified by heat 
before making the colorimetric determination. After alkali has been 
added to the medium, the reaction should be determined again as a con- 
trol on the accuracy of the work. 

1 It is stated by Hurwitz, Meyer and Osteoberg that the amount of normal alkali 
to be added cannot be calculated accurately in this way because of certain variables, 
i. e., alteration in volume because of the addition of concentrated rather than dilute 
alkali, the difference in dilution of the "buffers" by alkali solutions of different strengths, 
and the possible lack of correspondence between n and ^ solutions. They recommend 
plotting a curve from the data obtained by the titration of average laboratory samples. 
The curve is constructed in the following manner: Add to 25 cc. portions from each 
of the two media, one originally acid and one alkaline in reaction, graduated amounts 
of |* alkali and acid respectively in amounts of 0.1 cc. to the first portion, 0.2 cc. to 
the second, and so on up to 0.5 cc. to the last. From each of these portions, take 3 
cc. and add 0.3 cc. of the phenol-red indicator solution. These serve as standards. 
Take another 3 cc. sample of the medium to which neither acid nor alkali has been 
added and add 0.3 cc. of indicator solution, titrating with ^ alkali or acid until the 
color matches successively that of each of the five tubes containing normal alkali or 
acid. Plot the amounts of ^ solution required per 3 cc. as abscissae and the correspond- 
ing amounts of ^ solution per liter as ordinates. With the curve which was plotted by 
Hurwitz et al., if 0.46 cc. of ^- alkali were used in titration to secure the desired pH, then 
9 cc. of n alkali solution should be added to a liter of medium. After the curve has 
been prepared for a given set of solutions, titration of a 3 cc. portion may be carried 
and the amount of 5 solution necessary to adjust the reaction of one liter may be read 
directly from the curve. (See Fig. 135.) 



420 



BACTERIOLOGICAL METHODS 



Clearing Media.— This is accomplished by the coagulation of dis- 
solved proteins by heat, the coagulum enmeshing fine particles. When 
medium is devoid of protein, white of eggs must be added. The white 
of one or two eggs is beaten with about 20 cc. of water and stirred into 



24 





t 


1 


7 


f 




J 


f 


r 


7 : 


/ 


r 


i 


■J~ 


7' 


_p£ 


' xT 


-&- ^ 


^t- ^ 


/ 7 


/ -A*r- 


M 


. /■ a* 


A /'~ : ' 


1 / 


// 


~P 


/ 


/ I 



0.1 o.: 



0.4 0.5 0.C 0.7 0.8 0.0 1.0 



L.2 1.3 1.1 1. 



Fig. 135. — Curves indicating the actual amounts 
adjust 1 liter of medium to the desired hydrogen-ion 

tin- amounl of N (i acid "i alkali used. (Johns Hopkins 



of ~ acid or alkali necessary to 
concentration, as indicated by 
Hospital Bulletin.) 



PREPARATION OF MEDIA 



421 



the medium, which should be cooled to about 55° C. It is then heated 
in the Arnold sterilizer for forty-five minutes, when it is taken out, 
thoroughly shaken to break up the coagulum, and again heated in the 
Arnold sterilizer for fifteen minutes. This is referred to in the following 
directions as "clearing with egg." After coagulation, the medium is 
filtered. 

Filtering.— Fluid media may be cleared by filtration through close 
filter-paper. It is advisable to plait the paper or to use a corrugated 
funnel. Should coagulum be present, cotton should be employed. A 
spiral of wire is placed in the bottom of a large glass funnel and across 







vy 



Blood J 

Air J 

Mark *U 

Bacteria — *M 



Fig. 136. — A, Hopkin's tube; B, Wright's pipette. 

it are laid at right angles two long thin strips of absorbent cotton. 
The ends are made to adhere to the sides of the funnel by moistening 
them with water. The medium is run in along a glass rod to direct 
the stream so that it will not break through the filter. If it be cloudy 
as it first comes through it should be poured back until the pores are 
filled with coagulum. The funnel should be heated before use to 
prevent solidification. A hot-w.ater jacket, fitting around the funnel, 
is most convenient. 

Tubing.— After medium has been cleared and filtered, it is liquified 
by heat and poured into a glass funnel. Rubber tubing should be 
attached to the end of the funnel and a pinch-cock used to control 



422 BACTERIOLOGICAL METHODS 

discharge (Fig. 133,/). The medium is run into tubes,. placing about 
5 to 6 ce. in each tube. The tubes are stoppered and sterilized. 

Sterilization. — Hot Air. — All glassware and metal instruments may 
be sterilized in an oven or hot air sterilizer, preferably one of the 
Lautenschlager type for one hour at 150° C. Flasks, bottles, and 
tubes should be plugged with raw cotton. Petri dishes may be placed 
in a cylindrical copper box or may be wrapped with paper in sets of 
two or three. Pipettes may be wrapped in paper. One exposure to 
this temperature kills both spores and bacteria. 

Steam. — Steam is usually used for sterilization of media. In the 
Arnold sterilizer, the steam rises to the top and condenses, the water 
falling down the wall to the vessel below where it is boiled. Two 
types are available, one in which a cover is lifted off and the other, with 
a door on the side, Boston Board of Health type. Sterilization with 
this apparatus is conducted on the fractional plan, that is, at a tempera- 
cure of 100° C. for twenty minutes on each of three successive days. 
This is necessary since a single sterilization at 100° C. kills the bacteria 
but not the spores. The spores develop during the time between 
sterilizations and are destroyed by the subsequent heating. Media 
containing sugar are usually sterilized in the Arnold rather than in the 
autoclave to avoid the possibility of breaking down sugar. Although 
an autoclave is expensive and not absolutely necessary, it is very 
convenient. The higher temperatures which can be attained kill all 
spores so that it is possible to complete the sterilization with one 
exposure. 

Media in test-tubes are subjected usually to 15 pounds pressure 
(121.3° C.) for fifteen minutes. Flasks containing larger quantities 
of fluid should be left in for a longer period of time, from thirty to sixty 
minutes, depending on the amount of fluid to be sterilized. The 
temperature varies with the pressure, the equivalents being shown in 
the following table, taken from Hiss and Zinsser: 

1 pound of pressure equivalent to temperature of 102.3° C. 
5 pounds of pressure equivalent to temperature of 108.8° C. 

10 pounds of pressure equivalent to temperature of 115.°6 C. 

15 pounds of pressure equivalent to temperature of 121.3° C. 

20 pounds of pressure equivalent to temperature of 126.2° C. 

30 pounds of pressure equivalent to temperature of 134.6° C. 

Cotton, gauze, and glass hypodermic instruments may be sterilized 
either by hot air or steam, although the latter is preferable since it 
obviates the possibility of damage due to excessive heat. 

Slanting Solid Media.— Solid medium is usually slanted after 
sterilization. As soon as the tubes are removed from the sterilizer, 
they are laid in a nearly horizontal position, the upper ends being 
raised slightly by supporting them on a length of glass tubing or a flat 
strip of wood. The medium should not be allowed to come in contact 
with the stoppers. Agar should be left in this position for at least 
twenty-four hours. 



FORMULM FOR MEDIA 423 

Storing Media.— Media should be kept in the refrigerator. When 
only small quantities are used at a time, the bulk may be kept in 
bottles or flasks, the contents of which may be melted, tubed and 
resterilized in small lots as needed. Evaporation may be prevented 
by covering the cotton plugs with rubber caps or tin-foil, or by pushing 
in the plugs and by stoppering the tubes with corks or melted paraffin. 

FORMULA FOR MEDIA. 

. The Standard Methods of the American Public Health Association 
gives the following requirements for the materials used in making 
media. Liebig's meat extract and either Armour's, the Digestive 
Ferments Company's, or Fairchild's peptone should be used. Dis- 
tilled water should be employed. The sugars should be of the 
highest purity obtainable. The agar must be of the best quality 
and should be dried one-half hour at 105° C. before weighing. Since 
many brands contain a large quantity of sea salts, it is well to soak the 
agar in water and drain before use. Gelatin should be of light color, 
containing only a trace of arsenic, copper, and sulphides, and of such 
melting-point that a 10 per cent, solution will melt at 25° C. or over. 
It must be dried for one-half hour at 105° C. before weighing. Reagent 
litmus of the highest purity or azolitmin should be used, not litmus 
cubes. All chemicals should be of the highest purity. 
Meat Extract Broth (bouillon.) . 

Meat extract 5 gm. 

Peptone 10 " 

Sodium chloride 5 " 

Water 1000 cc. 

Put ingredients in an agate or enamelled pan, and weigh vessel. 
Heat over a flame until dissolved, stirring constantly. Make up lost 
weight by adding water. Titrate and adjust to proper reaction. Heat 
over free flame for five minutes. Sterilize and filter. If medium does 
not clarify completely, clear with egg. 

Meat Infusion Broth (bouillon.) . 

Fresh meat infusion. 

Lean beef or veal 500 gm. 

Water 1000 cc. 

Secure about one and a quarter pounds of meat, free from fat and 
tendinous material and force through the meat grinder twice. Place 
meat and water in an agate pan in the ice-chest over night. Strain 
through cheese-cloth. Add water to restore volume to 1000 cc. Use 
this in making the broth. 

Meat infusion broth. 

Fresh meat infusion 1000 cc. 

Sodium chloride 5 gm. 

Peptone 10 " 

Weigh the vessel containing the mixture. Warm over a flame to 
dissolve peptone, but do not allow the temperature to rise above 50° C. 



424 BACTERIOLOGICAL METHODS 

Restore lost weight by adding water. Determine reaction and adjust 
to neutral. Heat in Arnold sterilizer for thirty minutes. Shake well 
and heat again for fifteen minutes. Weigh and restore lost weight with 
water. Titrate and adjust to desired reaction. Heat if acid or alkali 
be added. Filter through cotton, tube, and sterilize. 

Sugar -free Broth.— Prepare a meat-infusion broth up to the point 
following heating in the Arnold sterilizer. Do not adjust the reaction 
but filter through cheese-cloth and bring up to original volume with 
water. Cool to room temperature and to each liter of medium add a 
tube of twenty-four-hour growth of B. coli communis. Stopper flask 
with cotton and place in the incubator for twenty-four hours. Sterilize 
in the autoclave at 15 to 18 pounds pressure for one hour. Restore 
volume with water. Adjust reaction to neutral. Heat in Arnold 
sterilizer for thirty minutes, filter through a double layer of filter paper, 
tube and sterilize. 

Carbohydrate Media.— These are usually made with sugar-free broth 
as a basis. If beef-extract broth be used, however, removal of carbo- 
hydrate is unnecessary. Carbohydrates are generally used in 1.0 per 
cent, concentration, that is, 1.0 gm. is used for each 100 cc. of media, 
and 1 cc. of saturated aqueous solution of neutral red, is added. The 
media should be heated in the Arnold sterilizer for twenty minutes on 
three successive days. 

If desired, a 10 per cent, solution may be made of most of the carbo- 
hydrates. Sterilize this at 100° C. for one and a half hours, and add 
10 cc. with a sterile pipette to each 100 cc. of sterile media. Tube 
mixture and sterilize at 100° C. for thirty minutes. Incubate tubes at 
37° C. for twenty-four hours to test sterility. 

Dunham's Peptone Solution. 

Sodium chloride 5 mg. 

Peptone 10 gm. 

Water 1000 cc. 

Dissolve by heating and filter. Adjustment of reaction is not 
required. 

Calcium Carbonate Broth. 

Calcium carbonate (powdered) lOgm. 

Nutrient broth 1000 cc. 

Tube and sterilize. 

Litmus Milk.— Steam fresh milk in an Arnold sterilizer for fifteen 
minutes and place in the ice-box over night. The cream rises to the 
surface. Siphon off the lower layer of fat-free milk and add sufficient 
litmus solution to give a deep lavender color. A solution of azolitmin 
is preferable. Tube, placing about 10 cc. in each tube. Sterilize in 
the Arnold sterilizer for twenty minutes on three successive days. 

Hiss' Inulin Serum -water.— Dissolve 10 gm. inulin in 750 cc. of 
water and sterilize in the autoclave for fifteen minutes at 15 pounds 
pressure. Add 250 cc. of clear beef serum and enough aqueous litmus 



FORMULA FOR MEDIA 425 

solution to give a deep lavender. Mix thoroughly, tube with about 10 
cc. in each tube, and sterilize in the Arnold sterilizer for twenty minutes 
on three successive days. (For method of obtaining beef-serum, see 
Loeffler's Blood Serum.) 
Meat Extract Gelatin. 

Meat extract 5 gm. 

Peptone 10 " 

Sodium chloride 5 " 

Gelatin 120 " 

Water 1000 cc. 

Weigh mixture with containing vessel and dissolve ingredients by 
warming. Restore lost weight with water and adjust reaction to 
desired point. Cool to 60° C. and clear with egg. Heat in Arnold 
sterilizer for thirty minutes, shake, heat again for fifteen minutes, 
restore lost weight with water, and filter through cotton. Tube and 
sterilize in Arnold sterilizer for thirty minutes on three successive days. 

Me at -infusion Gelatin.— Make meat-infusion broth as directed, 
adding 120 gm. of gelatin, mixing all ingredients with water at same 
time. Carry through other steps as outlined, tube in sterile test-tubes, 
and sterilize in Arnold sterilizer for thirty minutes on three successive 
days. Longer sterilization may interfere with solidification of the media. 

Meat Extract Agar. 

Meat extract 5 gm. 

Peptone 10 " 

Sodium chloride 5 " 

Agar 15 " 

Water 1000 cc. 

Mix in an agate pan. Weigh vessel with contents. Heat over a 
free flame until agar is dissolved. This may require thirty to forty-five 
minutes. Restore weight with water. Determine reaction and adjust 
to desired point. Cool to 60° C. and add whites of two eggs, thoroughly 
beaten with a little water. Mix well. Heat in Arnold sterilizer for 
thirty minutes, shake or stir thoroughly, and heat again for fifteen 
minutes. Restore lost weight with water. Titrate and again adjust 
reaction if necessary. Filter through cotton, tube, and sterilize. 

Meat-infusion Agar. 

Double-strength Meat-infusion. 

Lean beef or veal 500 gm. 

Water 500 cc. 

Secure about one and one quarter pounds of meat free from fat and 
tendinous material' and run through the meat grinder twice. Place 
the meat and water in an agate pan in the ice-chest over night and 
strain through cheese-cloth in the morning. Add water to restore the 
volume to 500 cc. Use this in making broth as indicated below. 

Meat^infusion Agar. 

Peptone 10 gm. 

Sodium chloride 6 " 

Double-strength meat-infusion 500 cc. 



426 BACTERIOLOGICAL METHODS 

Add peptone and salt, weight the vessel, warm over free flame to 
dissolve, keeping the temperature below 50° C. Restore lost volume 
with water, titrate, and neutralize. 

Dissolve 15 gm. agar in 600 cc. water by heating over a free flame 
for thirty to forty-five minutes. Cool to about 50° C. and add to the 
meat-infusion broth. Weigh the mixture. Heat in Arnold sterilizer 
for thirty minutes, shake thoroughly, heat again for fifteen minutes, 
and adjust to former weight by adding water. Determine volume, 
titrate, and adjust reaction to desired point. Boil over free flame for 
two minutes, stirring constantly. Filter through cotton, passing back 
through same filter until clear. Tube and sterilize. 

Glucose Agar. — Prepare agar by one of the preceding methods. 
Add 10 gm. of glucose to 1000 cc. of filtered clarified agar. Tube and 
sterilize in the Arnold sterilizer for twenty minutes on three successive 
days. 

Avery's Blood Oleate Agar.— See page 360. 

Blood Agar.— See pages 359 and 362. 

Agar and Broth for Pneumococcus Culture.— See page 362. 

Hydrocele or Ascitic Fluid Agar.— Obtain hydrocele or ascitic fluid 
under aseptic conditions. Melt agar by placing the tubes in boiling 
water. Reduce temperature of water to 50° C. and with a sterile 
pipette add to each tube 1 or 2 cc. of body fluid. Allow tubes to 
solidify in slanting position. 

Thomson's Medium for Gonococcus.— Prepare 2.5 per cent, nutrient 
agar in the usual way, using 1 per cent, peptone. Adjust reaction to 
+0.6 acid. Instead of adding sodium chloride only, add salts employed 
in Ringer's solution, namely, 9 gm. of sodium chloride, 0.25 gm. of cal- 
cium chloride, 0.42 gm. of potassium chloride to each liter of medium. 
Add 2.5 per cent, of glucose. Clear and filter agar, place 4 cc. in tubes, 
and sterilize. When ready to add the human serum, melt agar, cool 
to about 50° C.j and add 1 cc. of human serum to each tube. Mix 
the contents of the tube thoroughly by rolling the tube between the 
palms, and allow to solidify in the slanting position. 

Human serum is readily obtained by venipuncture (see page 126). 
drawing blood into a sterile tube containing a few crystals of sodium 
citrate. Centrifugalize blood, remove serum with a sterile pipette and 
add to the agar tubes. Thomson states that taking blood from an 
individual under mercury or arsenic treatment does not interfere with 
growth of the gonococcus. 

Petroff's Medium.— See page 358. 

Loeffler's Blood Serum.— Obtain beef blood from the slaughter-house 
in a large enamel-ware pail. Care should be taken to keep the blood 
free from hair and other foreign substances. Separate the clot from the 
side of the vessel with a glass rod and place the pail in the ice-chest 
over night. In the morning, remove the serum with a pipette. If it 
be cloudy, put it in a large centrifuge tubes and throw down the cells. 

A 1 per cent, glucose meat-infusion broth is required. The reaction 



FORMULA FOR MEDIA 427 

should be made neutral to phenolphthalein. Mix 3 parts of serum with 
1 part of dextrose broth, and tube in small tubes. Place tubes in a 
slanting position in Koch serum-coagulator (inspissator) and heat 
gradually until the temperature reaches 90° C. Keep temperature at 
this point until the medium has coagulated completely. It is impor- 
tant that the temperature be raised slowly to prevent formation of 
bubbles in the media. Sterilize on the second and third days in the 
Arnold sterilizer, raising temperature slowly to the boiling-point and 
maintaining it at this point for twenty minutes. , 

In the absence of an inspissator, the medium may be solidified in the 
autoclave by raising the temperature very gradually to 105° C. or in 
the Arnold sterilizer by covering the top with a cloth instead of the 
usual cover. 

Dorset's Egg Medium.— Wash eggs carefully with water and then 
with 5 per cent, phenol solution. Break them and pour contents into 
a sterile Erlenmeyer flask. Add 25 cc. of water for every 4 eggs. 
Mix and strain through sterile cheese-cloth. Tube and inspissate as 
with Loeffler's blood serum. 

Potato.— Scrub large potatoes under running water, and cut cylinders 
with an apple corer. Remove the skin from both ends and divide the 
cylinders with an oblique cut to form wedge-shaped pieces. Wash 
them in running water for several hours and place in tubes with the 
large end down. Sterilize in Arnold sterilizer for twenty minutes on 
three successive days. 

Endo's Fuchsin Agar.— (Standard Methods, American Public Health 
Association.) 

Beef extract , 5 gm. 

Peptone 10 " 

Agar 30 " 

Water 1000 ce. 

Place in flask, weigh flask and contents, and boil on water-bath until 
the agar is dissolved. Make up loss of weight by adding water. Cool 
mixture to 45° C. in cool water-bath and then warm to 65° C. in same 
bath without stirring. Make up lost weight, titrate, and if necessary 
adjust reaction to between neutral and +1. Filter through cotton 
until clear. Distribute between flasks in 100 cc. quantities. Sterilize 
in autoclave for fifteen minutes at 15 pounds pressure. Make a 10 
per cent, solution of basic fuchsin with 95 per cent, alcohol. After 
this has stood for twenty hours, decant the supernatant fluid and keep 
as a stock solution. 

When ready to make plates, melt a flask containing 100 cc. of agar 
in the Arnold sterilizer or on a water-bath. Dissolve 1 gm. of lactose 
in 15 cc. of distilled water, with heat if necessary, and 0.25 gm. anhy- 
drous sodium sulphite in 10 cc. water. Add 0.5 cc. of the stock fuchsin 
solution to this solution of sodium sulphite and then mix the fuchsin- 
sulphite solution with the lactose solution. Add the resulting mixture 



428 BACTERIOLOGICAL METHODS 

to the melted agar. The sulphite solution must be fresh. Plates are 
poured and allowed to harden in the incubator before use. On this 
media typhoid colonies appear as clear, colorless dew-drops while 
typical colon colonies are colored red with fuchsin. 

Russell's Double Sugar Agar.— Prepare plain agar, making the con- 
centration of agar 2 to 3 per cent. The reaction should be about +0.7. 
Add 5 per cent, aqueous solution of litmus to produce a distinct purple 
color. Then add normal sodium hydrate until the mixture is neutral 
to litmus. Add dextrose to make a concentration of 0.1 per cent, 
and lactose to make a concentration of 1 per cent. Before adding 
dissolve sugar in a small quantity of water. Tube and sterilize in the 
Arnold sterilizer for twenty minutes on three successive days. Slant 
the tubes and store in a dark Dlace. 

Inoculate the tube by stroking the surface and stabbing into the butt. 
After incubating from eight to eighteen hours, the typhoid bacillus 
surface growth is filamentous and colorless on a blue background, 
while that in the lower part of the butt is a deep red. In the imperfect 
anaerobic conditions in the butt, the organism obtains its oxygen by 
breaking down dextrose with the formation of acid. No acid is formed 
in the presence of oxygen. The colon bacillus shows abundant gas 
and acid formation, the medium being reddened throughout. The 
paratyphoids have a surface growth like typhoid while the butt shows 
reddening and a few small gas bubbles. 

Levine's Simplified Eosine-methylene-blue Agar.— 

Agar 15 gm. 

Peptone 10 " 

Dipotassium phosphate 2 " 

Water 1000 cc. 

Boil the ingredients until dissolved and make up the loss of water 
due to evaporation. Place measured quantities into bottles or flasks 
and sterilize in autoclave at 15 pounds pressure for from fifteen to 
twenty minutes. 

Just before using, the agar is melted and to each cubic centimeter 
is added the following: 

Lactose (20 per cent, solution) 5 cc. 

Eosine, yellow (2 per cent, aqueous solution) 2 " 

Methylene blue (0.5 per cent, aqueous solution) 2 " 

It is desirable to have the last three solutions sterilized in the 
autoclave before adding to medium. 

After agar has been mixed, pour into plates and allow to harden. 
Inoculate by streaking surface. Colonies of typhoid bacilli are white, 
those of colon are blue. 

MacConkey's Bile -salt Agar. 

Sodium glycocholate 8 gm. 

Peptone 20 " 

Lactose 10 " 

Agar 15 " 

Water, to make 1000 cc. 



FORMULA FOR MEDIA 429 

Dissolve the agar and peptone in the water, filter, and clear. Add 
the lactose and sodium glycocholate, tube and sterilize in the Arnold 
sterilizer on three successive days for twenty minutes. 

Colonies of B. typhosus produce no change while those of B. coli 
produce acid and cause precipitation of bile salts. 

Krumwiede's Brilliant Green Agar. 

Beef extract 3 gm. 

Sodium chloride 5 " 

Peptone 10 " 

Agar 15 " 

Water 1000 cc. 

Dissolve in autoclave, clear and filter. Add alkali to make slightly 
alkaline to litmus, bottle in 100 cc. quantities, and sterilize. 

When medium is to be used, melt 100 cc. and add 1 cc. of Andrade's 
indicator. Adjust the reaction to the neutral point to the indicator, 
that is, until the red color of the hot medium fades completely on 
cooling. The reaction point is best determined by titration of a small 
quantity. 1 

Add 5 cc. 20 per cent, sterile lactose solution, 5 cc. of 0.1 per cent, 
sterile glucose solution, and brilliant green solution, either 0.2 cc. or 
0.3 cc. of 0.1 per cent, dilution per 100 cc. of melted agar. The medium 
is poured into 6 Petri dishes to give thick plates. The plates are left 
uncovered until dry. Any variation in the composition of the medium 
requires a readjustment of dye concentration. Brilliant green inhibits 
the growth of all Gram-positive and many Gram-negative cocci, and 
shows differential action with colon-typhoid group, colon bacilli being 
extremely susceptible to the dye, while typhoid bacilli grow as a snow 
flake colony with delicate serrated edge, which may be slightly tinged 
with red from acid production. 

Conradi's Bile Medium. 

Peptone 20 gm. 

Glycerol 100 cc. 

Ox-bile 900 " 

Ox-bile is obtained from the abattoir; glycerol and peptone are 
added; the mixture is tubed and sterilized in the Arnold sterilizer for 
twenty minutes on three successive days. 

Jackson's Lactose-bile Medium. 

Peptone 10 gm. 

Lactose 10 " 

Ox-bile 1000 cc. 

The medium is tubed in fermentation tubes and sterilized in the 
Arnold sterilizer for twenty minutes each on three successive days. 

Robertson's Cooked Meat Medium for Anaerobes.— Mince 8 ounces of 
bullock's heart very fine and grind in a mortar. Add 8 ounces of water 
and heat slowly, to cook the meat thoroughly. Add normal sodium 
hydrate until the mixture is alkaline to litmus. Divide into tubes 
and autoclave. 

1 Andrade's indicator is prepared byjadding 16 cc. normal sodium hydrate to 0.5 per 
cent, aqueous solution acid'fuchsin. The dye is ^alkalinized slowly and (turns red^in 
the presence of acid. 



APPENDIX. 



EXAMINATION OF A LARGE NUMBER OF URINE SPECIMENS 
IN A HOSPITAL LABORATORY. 

The standing orders of most well-organized hospitals prescribe a 
certain routine, such as the submission of first specimens immediately 
upon admission, of daily and weekly specimens, and of preoperative 
and postoperative specimens in surgical cases. The examination of 
preoperative specimens is of especial importance, since the surgeon's 
choice of the anesthetic is often based upon the report. When twenty- 
four-hour specimens are desired they should be collected in the wards 
and placed in large containers, in which the entire specimen may be 
sent to the laboratory, where the quantity may be measured accurately 
by the laboratory worker. One of the greatest difficulties in having 
reliable urinalyses made in hospitals is the extreme difficulty that is 
encountered in training orderlies and even nurses in the importance of 
the saving of all the urine. 

The specimen should be sent to the laboratory properly labelled. 
The label should bear the name of the patient, the name of the attend- 
ing or house physician, a notation as to whether it be a single or a 
twenty-four-hour specimen, the ward or room number, and the date. 

Nurses should be instructed to have urine voided and sent to the 
laboratory only in thoroughly cleaned vessels, and to send only speci- 
mens, free from admixture with fecal material. 

In most hospitals it is the practice to send the specimens from the 
different wards to the laboratory in the morning. The person whose 
duty it is to examine them is confronted with the problem of making a 
large number of examinations with as great an economy of time as is 
consistent with careful work. A definite routine is necessary. The 
specimens should be given working serial numbers, beginning each day 
with 1. This should be written on the label which accompanies the 
specimen. A wooden test-tube rack is convenient. This should be 
made with five rows of holes and the holes in the front row should be 
numbered serially, beginning with 1 . The holes in the front row should 
be large enough to receive tubes 2.5 by 18 cm. while those in the other 
four rows need only be large enough to receive test-tubes of the ordinary 
size. The large tubes in the front row serve to float the hydrometer. 
Before beginning the tests, one row of tubes should be charged with 
Benedict's solution (5 cc. in each tube) and one with nitric acid (5 cc, 



432 APPENDIX 

in each tube). The tubes in one row should be left empty, and the 
fifth row should be filled with 15 cc. centrifuge tubes, numbered serially 
to correspond with the numbers of the tubes in the front row. 

The specimens may be poured from the containers into the large 
test-tubes which bear corresponding numbers. These tubes serve as 
reservoirs and are convenient for taking the specific gravity. Small 
bits of red and blue litmus paper are dropped into the specimens. The 
hydrometer is moved from one large tube to the next, rinsing after 
each reading under running water. A hydrometer made after the 
principle of the battery tester (see Fig. 61 b), is serviceable. As the 
reaction and specific gravity are taken, they should be noted in the 
record. Before beginning the chemical examination, it is well to place 
samples of urine in the numbered centrifuge tubes and to allow centri- 
fugalization to proceed while the chemical work is being carried out. 
At the close of the chemical procedure, the centrifugalized specimens 
will be ready for microscopical examination. 

For albumin tests, enough of each specimen may be poured into the 
row of empty tubes to fill the tubes nearly to the top. These portions 
are used for the test by the heat and acetic acid method. A few cc. of 
each specimen are run over the nitric acid in the tubes which have been 
previously charged with this reagent for the cold nitric acid test. Into 
the row of tubes which have been charged with Benedict's solution there 
should be added 5 to 8 drops of urine, using a medicine dropper which 
may be rinsed with water after use with each specimen. If Fehling's 
solution be employed, the urine will be poured from the large test-tube 
into the corresponding test-tube containing the Fehling's. It is con- 
venient to have a water-bath for boiling all these tubes at one time. 
The rack of the water-bath should be prepared with holes to carry the 
desired size of tube and near each hole should be stamped a number to 
correspond with those on the wooden rack to prevent confusion of the 
tubes, according to the suggestion of Myers and Fine. The layer test 
may be performed by allowing the urine to run up into a piece of glass 
tubing of wide bore, followed by a small quantity of nitric acid, wash- 
ing the tubing out after the test with water. 

The results of examination may be recorded in a small pocket note- 
book, ruled with parallel columns in which may be recorded the names, 
wards, numbers of the specimens, and the findings. Tha date may be 
transcribed to the hospital records. The advantage of such a book is 
that it is convenient for making the immediate notes and it gives the 
person making the tests a record to which he may refer in case the report 
is lost or misplaced. 

PREPARATION OF NORMAL SOLUTIONS. 

Normal solutions are those which contain in 1 liter of water at 16° C. 
the hydrogen equivalent of the active reagent weighed in grams. The 
hydrogen^equivalent of a base, acid, or salt is the molecular weight 



PREPARATION OF NORMAL SOLUTIONS 433 

divided by the valence, the valence in the case of a base being shown 
by the number of hydroxyls combined with it, while that of acids is 
shown by the number of replaceable hydrogen atoms. For example, 
1000 cc. of a normal solution of hydrochloric acid would contain 36.47 
gm. of acid, since hydrochloric acid is monovalent, and the molecular 
weight is 36.47. Oxalic acid, however, is divalent, so one-half of its 
molecular weight is taken. 

The following solutions are those which are used most frequently in 
the laboratory. The table shows the amount of the substance which 
should be contained in a liter of solution. 

Grams 
per liter. 

Acetic acid 60.04 

Ammonia 17.03 

Ammonium sulphocyanate 76.12 

Hydrochloric acid 36.47 

Iodine 126.92 

Oxalic acid (COOH.COOH.2H 2 0) . . 63.03 

Potassium hydroxide 56.11 

Silver nitrate 169.89 

Sodium carbonate 53.01 

Sodium hydroxide 40.01 

Sulphuric acid 49.04 

Equal volumes of normal solution combine exactly with each other, 
that is, 10 cc. of normal sodium hydrate solution exactly neutralize 
10 cc. of normal hydrochloric acid solution. 

Seminormal, quintinormal, decinormal, vigintinormal, and centi- 
normal solutions are used, and are designated respectively by the 
abbreviations | or 0.5 N, f or 0.2 N, T N o or 0.1 N, & or 0.05 N, T ^ or 
0.01 N. They are usually prepared from corresponding normal solu- 
tions by dilution with water. For example, yjj- hydrochloric acid would 
be prepared from y hydrochloric acid by placing 100 cc. of the latter 
in a 1000 cc. volumetric flask, adding enough distilled water at 16° C. 
to make the volume 1000 cc. 

The solution most frequently required in the clinical laboratory is 
decinormal sodium hydrate. Unless accurate balances and volumetric 
glassware be available, it is better to secure this solution from a reliable 
chemist. 

Since it is impossible to weigh sodium hydrate accurately because its 
weight changes due to the absorption of water, a sodium hydrate 
solution must be made up approximately and then titrated against an 
acid of known strength. An acid which is available as a standard is 
oxalic acid. 

Preparation of Normal Oxalic Acid.— Perfect crystals of a chemically 
pure preparation should be selected, and 63.03 gm. weighed out on an 
analytical balance. The crystals are transferred to a volumetric flask 
and enough distilled water is added to dissolve. Distilled water is 
then added to bring the volume exactly to the 1000 cc. mark, the flask 
is stoppered, and the contents are mixed thoroughly. The accuracy 
28 



434 APPENDIX 

of this solution may be checked by titration against normal sodium 
carbonate. 

Preparation of Normal Sodium Hydrate. — "Sodium hydrate by 
alcohol" should be obtained. About 45 gm. of the sticks are weighed 
out and dissolved in a liter of distilled water. After the temperature 
has been reduced to 16° C, 50 cc. of the alkali solution are measured 
into a white porcelain dish. A drop of methyl orange solution is added 
and normal oxalic acid solution run in from a burette while stirring the 
mixture until the pink color becomes yellow. The reading of the 
burette is taken. If the sodium hydrate solution be of proper strength, 
50 cc. of oxalic acid should have been used to neutralize. As made up, 
the solution will probably be too strong, so that less than 50 cc. of acid 
will be required for neutralization. When this is the case, the amount 
of water which must be added to the sodium hydrate may be estimated. 
For example, when only 45 cc. of oxalic acid solution are required, the 
solution is 5 per cent, too strong, and 1 cc. of distilled water should be 
added for each 19 cc. of solution in the flask. After the water has been 
added, the solution should be titrated again and if it still be too strong, 
the process of dilution and titration should be repeated until exactly 
50 cc. of y oxalic acid solution are required to neutralize 50 cc. of 
sodium hydrate solution. Decinormal solution may be prepared 
from the normal solution by placing 100 cc. of the latter in a liter volu- 
metric flask and adding distilled water to bring the volume exactly to 
1000 cc. 

Preparation of Normal Sodium Carbonate.— Sutton 1 recommends the 
use of sodium carbonate for the preparation of the standard alkali 
solution against which the acid should be titrated. 

A platinum crucible is half filled with powdered sodium bicarbonate. 
The crucible is placed in an air-bath whose temperature is 200° C. The 
temperature is raised to about 270° C, not over 300° C. The crucible 
is allowed to remain at this temperature for about half an hour, when 
it is removed and the sodium carbonate cooled in an exsiccator. When 
cool, the powder is transferred to a cool and perfectly dry bottle from 
which it is removed and weighed rapidly. About 2 gm. are weighed 
out and dissolved in 80 or 100 cc. of water. A few drops of methyl 
orange are added to the solution and the acid to be standardized is 
added from a burette until the mixture has a pure rose color. The 
reading is taken. It should take 1Q0 cc. of a normal acid to neutralize 
5.3 gm. of sodium carbonate. The amount of acid (X) which should 
have been required to neutralize the amount of sodium carbonate used 
may be computed as follows: 

Gm. sodium carbonate: 5.3 : : cc. acid solution used : X. 

For example, if 2.4 gm. of carbonate were weighed out, and 42 cc. 
of acid solution were employed, then the equation would be: 

2.4 : 5.3 : : 42 : X = 92.7 
1 Volumetric Analysis, Ninth Edition, 1904, P. Blakiston's Son & Co., Philadelphia. 



STAINING METHODS AND PREPARATION OF STAINS 435 

This determination should be checked by weighing out a second 
quantity of sodium carbonate and repeating the titration, taking the 
average of the results. Should the average be 92.7, the acid solution 
is too strong, i. e., 92.7 cc. contains as much acid as 100 cc. should, 
so that 900 cc. of the solution should be measured in a 1000 cc. volu- 
metric flask, 27 cc. measured out with a burette, and water added to the 
liter mark. Sutton recommends preparing sulphuric acid solution 
standardized in this way against normal sodium carbonate solution. 
From the acid solution, a normal sodium hydrate solution may be made. 

STAINING METHODS AND PREPARATION OF STAINS. 

All of the ordinary stains are prepared very simply from the powdered 
dyes. It is much better for the worker to make his own stains from the 
dyes than to purchase them in the liquid form. Saturated alcoholic 
solutions may be kept on hand, and dilutions made from them as 
desired. The following table, taken from Wood, shows the amount of 
dye required to saturate 100 cc. of solvent: 

Fuchsin (to saturate 100 cc. of 96 per cent, alcohol) . . . 3.0 gm. 

Gentian violet (to saturate 100 cc. of 96 per cent, alcohol) . . 4.8 " 

Gentian violet (to saturate 100 cc. of water) 1.5 " 

Methylene blue (to saturate 100 cc. of 96 per cent, alcohol) . 2.0 " 

Methylene blue (to saturate 100 cc. of water) 6.7 " 

Wright's stain (to saturate 100 cc. of absolute methyl alcohol) 0.3 " 

The majority of the saturated alcoholic solutions keep indefinitely 
as stock solutions. Several manufacturers offer compressed tablets of 
powdered stains which may be dissolved in small quantities of diluent. 
These are convenient when stains are not used in large quantities. 

Loeffler's Methylene Blue Solution. 

Saturated alcoholic solution of methylene blue 30 cc. 

Potassium hydrate, 1 : 10,000 solution 100 " 

Films are stained from one to five minutes. Heat is usually unneces- 
sary but may be employed if more intense staining be desired. Diph- 
theria bacilli may be demonstrated by staining for five minutes and 
decolorizing with 0.5 per cent, acetic acid until the film has a gray tint. 

Carbol-fuchsin (Ziehl-Neelsen Solution). 

Fuchsin, saturated alcoholic solution 10 cc. 

Phenol, 5 per cent, aqueous solution 90 " 

Films are stained for about two minutes. The stain is heated to 
steaming but should not be allowed to dry. Fresh stain should be 
dropped on during the staining process. 

Pappenheim's Decolorizing Fluid. 

Rosolic acid (corallin) v 1 gm. 

Absolute alcohol 100 cc. 

Methylene blue to saturation, 

Glycerol 20 " 



436 APPEXDIX 

The glycerol is added after the other substances have been mixed. 

The decolorizing fluid is poured on a slide which has been stained 
with carbol-fuchsin and drained slowly, repeating the process 5 or 6 
times. 

Summary of Technic for Tubercle Staining. 

1. Cover slide with carbol-fuchsin and steam for two minutes. 

2. Wash off stain with water and place slide in acid alcohol (one part 
of hydrochloric acid and 99 parts of 85 per cent, alcohol) for three to 
five seconds, or until film shows only a faint pink color. 

3. Immerse in 95 per cent, alcohol until no further color is removed. 

4. Dry and stain with Loefner's methylene blue for two minutes. 

5. Dry and examine with oil immersion. 

As an alternative procedure, Pappenheim's rosolic acid decolorizing 
fluid may be applied after the slide has been stained with carbol-fuchsin, 
taking the place of steps, 2, 3, and 4. 

Niesser's Stain for the Diphtheria Bacillus. 

Methylene blue, in powder 1 gm. 

Alcohol, 96 per cent 20 cc. 

Acetic acid, glacial 50 " 

Distilled water 950 " 



The methylene blue is dissolved in the alcohol, and then water and 
glacial acetic acid are added, and the mixture filtered. 

Stain for from one to three seconds, wash in water, stain with Bis- 
marck-brown for five seconds or more, wash and dry. 

See also Loeffler's methylene blue above. 

Gram's Stain. — Aniline-water Gentian Violet.— One cc. of aniline 
oil is shaken up with 25 cc. of distilled water. The resulting emulsion 
is filtered through filter paper and used in making the stain as indicated 
in the formula : 

Watery emulsion of anilin oil 18 cc. 

Gentian violet, saturated alcoholic solution 2 " 

This solution keeps only a few days. It should be filtered before 
using. Films are stained from one to three minutes. 
Sterling's Gentian Violet Solution. 

Gentian violet 5 gm. 

Alcohol, 95 per cent 10 cc. 

Anilin oil 2 " 

Distilled water 88 " 



This solution has the advantage of lasting three or four months. 
The aniline oil and alcohol are thoroughly mixed by shaking and 
water is added. The gentian violet is ground up in a mortar mean- 



STAINING METHODS AND PREPARATION OF STAINS 437 

while adding the aniline solution slowly. When the maximum degree 
of solution has been attained, the resulting mixture is filtered. The 
stain is placed on the film and poured off. After one minute, the 
film is washed with Gram's iodine solution. It is covered with fresh 
iodine solution, which is allowed to remain for two minutes. 
Gram's Iodine Solution. 

Iodine 1 gm. 

Potassium iodide 2 " 

Distilled water 100 cc. 

The iodine and potassium iodide are dissolved by stirring them 
together in a beaker with 20 cc. of water, grinding the chemicals against 
the wall of the beaker with a glass rod until solution takes place, when 
the remainder of the water is added. 

This solution is used to wash off the gentian violet stain from the 
slide, when fresh solution is allowed to remain on the film for two 
minutes. It should be washed off in turn with absolute alcohol (98 
per cent.). The slide is then placed in absolute alcohol (98 per cent.) 
for from one to ten minutes, or until no further stain is removed. Water 
should not be used for washing the film at any stage of the staining 
process. The slide is now ready for counterstaining. 

Counterstains for Gram's Stain.— Bismarck Brown.— A saturated 
aqueous solution of Bismarck brown is made with the aid of heat. 
After cooling, it is filtered and diluted with 10 parts of water. The 
film may be stained from two to five minutes. There is little danger of 
overstaining. 

Diluted Carbol-fuchsin.— This should be made by diluting 1 part 
of carbol-fuchsin with 9 parts of distilled water. It is allowed to 
remain on the slide for about two minutes. 

Safranin.—A saturated solution of safranin is made with 95 per cent, 
alcohol. One part is diluted with 9 parts of distilled water. The stain 
is allowed to act for two minutes. 

Summary of Technic for Gram's Stain. 

1. Cover film with Sterling's gentian violet solution and allow to 
remain one minute. 

2. Wash off with Gram's iodine solution. 

3. Cover with fresh iodine solution and allow to remain two minutes. 

4. Wash off with absolute alcohol. 

5. Immerse in absolute alcohol from one to ten minutes, or until no 
further stain is removed. 

6. Dry and apply counterstain. 

7. Dry and examine with oil-immersion lens. 

Results with Gram's Stain.— Bacteria retaining the original stain and 
appearing purple or lilac are said to be Gram-positive while those that 
are decolorized, losing the gentian violet and taking the counterstain, 
are Gram-negative. 



438 APPENDIX 

( llassification of important pathogenic bacteria according to Gram's 
stain : 

Gram Positive (Retain the violet color) Gram Negative (Lose the violet color and take the 

counters tain) 

Bacillus aerogenes capsulatus Bacillus coli 

Bacillus anthracis Bacillus dysenterio? 

Bacillus botulinus Bacillus influenzae 

Bacillus diphtheria' Bacillus Koch-Weeks 

Bacillus subtilis Bacillus mallei 

Bacillus tetanus Bacillus Morax-Axenfeld 

Bacillus tuberculosis and other acid- Bacillus mucosus capsulatus 

fast bacilli Bacillus paratyphosus 

Diplococcus pneumoniae Bacillus pestis 

Micrococcus pyogenes Bacillus proteus 

Micrococcus pyogenes albus Bacillus pyocyaneus 

Micrococcus pyogenes aureus Bacillus typhosus 

Diplococcus gonorrhea 

Diplococcus intracellularis meningi- 
tidis 

Micrococcus catanhalis 

Spirillum cholera? 

Capsule Stains.— Hiss' Capsule Stain.— Preparations are made on 
slides or cover-slips, preferably from animal exudates. If preparations 
be made from cultures, a drop of fresh blood-serum should be added to 
the drop of culture, material in making the film. Water must not be 
used at any stage of the procedure. The film is dried in the air, care- 
fully fixed with heat, and flooded with a solution of gentian violet (5 cc. of 
saturated alcoholic solution diluted with 95 cc. of distilled water). The 
stain is steamed over a free flame for a few seconds, when it is washed 
off with a 20 per cent, solution of copper sulphate. This is poured off 
and the slide dried and mounted in balsam. The capsules appear as 
fainty blue haloes around dark purple cell-bodies. Avery and co- 
workers state that better results may be obtained by omitting heat 
fixation altogether, and by washing off the gentian violet solution with 
copper sulphate as soon as it begins to steam. 

Welch's Capsule Stain.— A cover-slip preparation, made in the usual 
way, is allowed to dry in the air without the aid of heat, and covered 
with glacial acetic acid for a few seconds. The acid is drained and the 
film covered with anilin-water gentian violet. Tins is poured off and 
replaced by fresh stain, repeating the process several times. The stain 
is left on for three or four minutes the last time and then washed off in 
2 per cent, sodium chloride solution. The cover-slip is mounted without 
drying on a slide in a few drops of the salt solution and examined with 
the oil-immersion lens. Water should not be used in making the pre- 
paration or during the process of staining. 

Staining for Spores.— Special methods are necessary to penetrate the 
resistant spore membrane. The film may be stained by steaming with 
carbol-fuchsin for two minutes, washing with water, and then decoloriz- 
ing with a 1 per cent, dilution of sulphuric acid in water. Counter- 
staining is accomplished with Loeffler's methylene blue. This is merely 



STAINING METHODS AND PREPARATION OF STAINS 439 

an adaptation of the method used for tubercle bacilli. The bodies of 
the bacteria stain blue, the spores are red. If the spores are too 
resistant to take up the stain with this method, either Moeller's or 
Huntoon's method may be employed. 

Moeller's Method. — A thin film is dried, fixed with heat, washed in 
chloroform for two minutes, then in water, and placed in 5 per cent, 
chromic acid for three minutes. This is washed off with water and the 
film covered with carbol-fuchsin, which is steamed for two minutes 
and washed off with water. Five per cent, sulphuric acid is used for 
decolorizing and methylene blue applied for two minutes as a counter- 
stain. The spores are red and the bodies of the bacteria blue. 

Huntoon's Method.— Two solutions are needed. 

A. 

Acid fuchsin 4 gm. 

Acetic acid, 2 per cent, aqueous solution 50 cc. 

B. 

Methylene blue , 2 gm. 

Acetic acid, 2 per cent, aqueous solution 50 cc. 

The two solutions are mixed and allowed to stand for fifteen minutes. 
The heavy precipitate which ensues is removed by filtration and the 
filtrate used for staining. The stain keeps for several weeks. 

For staining, a rather thick film is covered with the stain for one 
minute. The bright-red film is dipped in a dilute solution of sodium 
carbonate (7 or 8 drops of a saturated solution in 250 cc. of water) until 
it turns blue, when it is removed, washed with water, dried, and 
examined with the oil-immersion lens. The spores stain red and the 
bodies of the bacilli blue. 

Staining Flagella. — (Bunge's modification of Loeffler's method).— 
The procedure is difficult and requires strict adherence to detail. 
Cultures must be employed. To 2 or 3 cc. of distilled water in a test- 
tube is added enough growth from a young (twelve to eighteen hours) 
agar culture to produce slight cloudiness. The suspension may be placed 
in the incubator at 37° C. for a few hours to permit some development. 
A small drop of the suspension is spread upon a thoroughly cleaned 
cover-slip as a very thin film in order to secure quick drying. 1 The 
film is fixed by passing the cover-slip through the flame three times, 
and then covered with the mordant, which is allowed to act without 
heat for five minutes when it is warmed for one minute and washed off. 
After drying, it is stained with anilin-water gentian violet, warming 
gently for about a minute. The staining process may have to be 
repeated a number of times. The mordant is prepared according to the 
following formula: 

Tannin, saturated water solution 3 parts 

Ferric chloride, 25 per cent, watery solution 1 part 

1 See method for cleaning cover-slips described for making blood films, page 44. 



440 APPENDIX 

This must be allowed to stand several weeks before using. 

Staining Treponemata. — Methods of demonstrating treponemata by 
staining and dark-field illumination have been described on page 397. 

Wright's Stain.— The technic for staining with "Wright's stain has 
been given fully in the chapter on the Examination of Blood. The 
stain may be prepared from the powder by dissolving 0.1 gm. in 60 cc. 
of methyl alcohol (preferably Merck's reagent). The powder should 
be rubbed up in a mortar with pestle to ensure thorough solution. It 
should be filtered. It is important to have all apparatus free from acid . 

The stain may be prepared from methylene blue and eosin as follows: 

Methylene blue (B. X. or medicinal) is added to 0.5 solution of 
sodium bicarbonate in the proportion of 1 gm. of dye to each 100 cc. 
of solution. The mixture is placed in a flask or flasks so that the 
layers of fluid will not be more than 6 cm. deep and heated in a steam 
sterilizer at 100° C. for a full hour. It is then cooled by placing the 
flasks in cold water when the precipitate may be removed by filtration. 
•When cool, the solution should have a deep purple-red color when a 
thin layer is viewed with transmitted yellow artificial light. 

To each 100 cc. of the filtrate are added 500 cc. of a 0.1 per cent, 
aqueous solution of "yellowish, water-soluble" eosin. After thorough 
mixing, the heavy precipitate which forms is collected on a filter and 
dried. The powder may be put away and dissolved with methyl 
alcohol as desired. The proportions are given above. 

Wilson's Stain.— Silver oxide is freshly precipitated by dissolving 
2 gm. silver nitrate in 15 cc. of distilled water and adding 260 cc. of 
freshly prepared solution of calcium hydrate. 1 The brown precipitate 
of silver oxide is collected on a coarse filter paper, washed with water, 
and dried at a temperature not exceeding 80° C, when it is used in 
preparing solution A. 

Two solutions are made: 

Solution A. 

Methylene blue 2 gm. 

Sodium bicarbonate 1 " 

Silver oxide 2 " 

Distilled water . 200 cc. 

Solution B. 

Eosin (yellow, water soluble) 1 gm. 

Distilled water 200 cc. 

Solution A is boiled slowly in a deep porcelain dish, stirring occa- 
sionally for twenty minutes when about 65 cc. are poured into a 
graduated cylinder, and an equal quantity of water added to the 
boiling solution. In twenty minutes about one-half the quantity of 

1 Calcium hydrate may be prepared by dissolving a small quantity of unslaked lime 
in 2 or 3 liters of water, stirring well, and allowing to settle, when the supernatant layer 
is siphoned off to remove the impurities and fresh water is added. The washing pro- 
cess is repeated 2 or 3 times and the fluid from the third washing used in preparing the 
silver oxide. 



STAINING METHODS AND PREPARATION OF STAINS 441 

fluid in the porcelain dish is again poured into the graduate and boiling 
continued for twenty minutes. The solution is added to the other two 
fractions in the graduate and enough distilled water added to bring the 
total volume to 200 cc. The solution is immediately filtered through 
coarse filter paper and Solution B added. The two fluids are mixed 
thoroughly and at the end of fifteen to thirty minutes the mixture is 
filtered through hard filter paper to secure the brownish-red precipitate. 
During filtration the sides of the filter should be washed down with 
normal sodium chloride. The precipitate may be dried in the air, by 
suction, or preferably in a hot air sterilizer at a temperature not in 
excess of 60 to 80° C. The powder is bluish-black in color with green 
luster. It should be kept in a dark bottle away from the light. To 
make staining fluid, 0.4 gm. of the powder are dissolved in 100 cc. 
absolute methyl alcohol. 

Staining is carried out as with Wright's stain. The film is dried in 
the air and covered with stain. After a minute, an equal quantity of 
water is added. The diluted stain is allowed to act for four minutes 
when it is washed off with water until the film shows a pinkish-brown 
or tan shade. The film is dried and mounted in balsam. 

Giemsa Stain. 

Azur II eosin 3.0 gm. 

Azur II 0.8 " 

Glycerol (C. P.) 250.0 cc. 

Methyl alcohol (H. P.) 250.0 " 

For staining, fresh dilution is prepared by adding 1 drop to 10 cc. of 
distilled water. The preparation is dried in the air, fixed with methyl 
alcohol for two minutes, and covered with diluted stain. At the end of 
ten or fifteen minutes, this is washed off. The film is dried and mounted 
in balsam. Staining may be intensified by adding 1 or 2 drops of 1 
per cent, sodium carbonate to 10 cc. of diluted stain. 

Jenner Stain. 

Eosin (Grubler's water soluble) 1.2 per cent, solution in distilled 

water 100 cc. 

Methylene blue (Grubler's medicinal) 1 per cent, solution in dis- 
tilled water 100 " 

The solutions are stirred together in a beaker. The mixture is 
allowed to stand twenty-four hours, when it is filtered through paper to 
secure the precipitate, which is washed by pouring distilled water 
through the filter until the filtrate shows only a slight blue color. 
The precipitate is then scraped from the filter and dried in the air, 
without heat. 0.5 gm. of dry powder are dissolved in 100 cc. of 
methyl alcohol (H. P.). A blood film, dried in the air, is covered with 
the stain. After one to three minutes, it is rinsed with distilled water 
until the film is pale pink. 

Antiformin.— Antiformin may be prepared according to this formula: 

Sodium hydrate 7.5 gm. 

Liquor sodae chlorinate (Labarraque's solution) . . . . 100.0 cc. 



442 APPEXDIX 

Liquor soda- chlorinatae may be made according to the following 
directions after the I . S. pharmacopoeia, although Stitt recommends 

doubling the quantities of sodium carbonate and chlorinated lime. 
Liquor Sodse Chlorinatae. 

Sodium carbonate (monohydrated) 70 gm. 

Chlorinated lime 100 " 

Water, to make 1000 " 

Dissolve sodium carbonate in 500 cc. of hot water, and add it to a 
previously prepared magma of chlorinated lime, made by rubbing up the 
latter with 500 cc. of water. Shake the mixture well and warm if it 
gelatinizes. Filter through a moistened muslin strainer, returning the 
first of the filtrate through the strainer. Make up to 1000 gm. Keep 
in a dark cool place in well stoppered bottles. 

PURIFICATION OF PICRIC ACID. 

Picric acid may not be chemically pure even when so labeled. The 
following method has been recommended for determining its purity: 
A saturated solution is made in water. Some of this is placed in the 
left hand cup of the colorimeter. Ten cc. are placed in a tube or 
graduate and to it is added 0.5 cc. of 10 per cent, sodium hydrate 
solution. This is allowed to stand for ten minutes and is then placed 
in the right hand cup of the colorimeter. The left hand cup should be 
placed at 20 mm. When the color is matched exactly, the reading of 
the right hand cup should not be less than 15 mm. In other words, 
the picric acid should not show more than 25 per cent, reaction. 

Purification of picric acid may be carried on according to the method 
of Folin and Doisy. Six hundred grams of the wet picric acid are 
transferred to a large beaker or a large porcelain casserole, holding not 
less than 4 liters. Boiling water is added until the beaker is nearly full. 
In this should be placed 200 cc. of 50 per cent, sodium hydrate. The 
mixture is stirred and heated gently, if this be necessary, to dissolve 
all of the acid. While the mixture is still hot, 200 gm. of sodium 
chloride should be stirred in slowly. The beaker is then cooled until the 
temperature is about 30° C, stirring occasionally. Then the contents 
are filtered through a large Buclmer funnel and the sediment is washed 
a few times with 5 per cent, sodium chloride. The sediment is then 
transferred to a large beaker which is filled with boiling water. When 
the picrate has been dissolved, 50 cc. of 10 per cent, sodium hydrate 
should be added with stirring, and then 100 gm. of sodium chloride. 
This is cooled to 30° C. with stirring, filtered and washed with 5 per 
cent, sodium chloride solution as before. The solution and precipita- 
tion of the sodium picrate is repeated twice more. For the last wash- 
ing, the pure water is used instead of the sodium chloride. 

The purified picrate is then dissolved in a large beaker with boiling 
distilled water and is filtered while still hot through a large folded filter, 
collecting the filtrate in a large flask. To the hot filtrate is added 100 



PREPARATION OF CREATININE ZINC CHLORIDE 443 

cc. concentrated sulphuric acid which lias been previously diluted with 
2 volumes of distilled water. The liberated picric acid will begin to 
crystallize almost at once. A beaker is placed over the mouth of the 
flask and the flask is cooled under running water to about 30° C. This 
is filtered through a Buchner filter and is washed free from sulphate 
with distilled water. 



PREPARATION OF CREATININE ZINC CHLORIDE. 

Creatinine zinc chloride may be readily prepared by Benedict's 
modification of Folin's method. To about 10 liters of undecomposed 
urine are added 180 gm. of picric acid. The acid should be dissolved 
first in boiling alcohol, using approximately 100 cc. of alcohol for each 
40 gm. of picric acid. This hot alcoholic solution of picric acid should 
be poured into the urine while the latter is being stirred vigorously. 
After the mixture has stood over night in a precipitating jar, the 
supernatant fluid may be siphoned off. The residue is poured upon a 
large Buchner funnel and is drained with suction. After draining, it 
should be washed once or twice with a cold, saturated solution of picric 
acid and sucked dry by the vacuum. The dry, or nearly dry, precipi- 
tate should be placed in a large mortar or evaporating dish with 
sufficient concentrated hydrochloric acid to form a moderately thin 
paste. The proper amount is usually 60 cc. of acid for 100 gm. of 
picrate. This mixture is stirred thoroughly with a pestle for from three to 
five minutes, when it is filtered with suction through a hard filter paper, 
washing the residue with twice enough water to cover it. The washing 
process should be repeated twice when the filtrate may be trans- 
ferred to a large flask and neutralized with an excess of solid magnesium 
oxide, the variety is known commercially as the heavy magnesium 
oxide. The oxide should be added in small portions. When the 
hydrochloric acid has been completely neutralized, the mixture turns 
a bright lemon-yellow. Litmus may be used to determine the end- 
point of neutralization. This mixture is filtered with suction and the 
precipitate is washed two or three times with water. The residue is 
discarded and the filtrate is treated with sufficient glacial acetic acid 
to strongly acidify it. A few cc. of acid are usually sufficient for this 
purpose. No attention need be paid to a precipitate which may form 
at this point. The solution should then be diluted with about 4 
volumes of 95 per cent, alcohol and filtered with suction after it has 
been allowed to stand for fifteen minutes. Filtration is necessary to 
remove the slight precipitate which then forms, composed chiefly of 
calcium sulphate. The filtrate finally obtained is treated with 30 per 
cent, solution of zinc chloride. Three or four cc. of zinc chloride solu- 
tion should be used to each liter of urine originally employed. The 
mixture is stirred and allowed to stand over night in a .cool place. A 
precipitate should form almost at once. After standing over night, 
the supernatant fluid is poured off and the precipitate, creatinine 



444 APPEXDIX 

zinc chloride, is collected on a Buchner funnel, washed with water, 
next with 50 per cent, alcohol, then with 95 per cent, alcohol, and 
finally dried. The substance obtained should be a nearly white cry- 
stalline powder. Standard creatinine solution may be conveniently 
prepared from the creatinine zinc chloride by weighing out 1.6106 gm. 
of the thoroughly dried salt and dissolving it in sufficient decinormal 
hydrochloric acid to make 1 liter. Such a solution contains 1 mg. of 
creatinine per cc. From this one may readily prepare stock solutions 
for the three standards for blood determination which will contain 0.3, 
0.5 and 0.1 mg. of creatinine per 100 cc. of saturated solution of picric 
acid by placing in 100 cc. volumetric flask 0.3 cc, 0.5 cc. and 1.0 cc. of 
the standard solution and bringing to volume with saturated solution 
of picric acid. The standard for determinations in the urine is pre- 
pared each time by placing 1 cc. of the standard (containing 1 mg. 
per cc.) in a 100 cc. volumetric flask, and adding 20 cc. of saturated 
solution of picric acid, 1.5 cc. of 10 per cent, sodium hydrate, and water 
to 100 cc. 

APPARATUS. 

The first list includes apparatus required to carry out the simple 
routine tests. That required for more complicated procedures, such 
as complement-fixation tests or blood chemistry, is given in special lists 
which follow. Mention of apparatus required for other less frequently 
employed methods will be found in the text. Bacteriological apparatus 
is described in the chapter on Bacteriological Methods. All the lists 
are subject to adaptations and substitutions. The number of pieces 
of the different articles has not been given, because this will depend 
entirely upon the number of specimens which are to be examined and 
the capacity of the laboratory. The reagents for all methods have been 
given with the description of the methods, since this seemed to be the 
more convenient arrangement. 

Minimum Equipment for Clinical Laboratory. 

Alcohol lamp. 

Bacteriological loop. 

Beakers, about 50 cc. and 100 cc. capacity, with lip. 

Blood lancet. 

Bottles, one ounce capacity, to contain solutions for blood counting, 
i. e., alcohol, ether, distilled water, Hayem's solution, 0.5 per cent, 
acetic acid, f HC1. 

Burner, Bunsen, Tyrrill, or micro type. 

Burette, 25 or 50 cc. capacity, graduated in 0.1 cc, with glass stop- 
cock or rubber tubing outlet and Mohr pinchcock. 

Burette stand for supporting burette. 

Camel's-hair brush. 

Centrifuge, water, hand, or electric power. The last form is pre- 
ferable because cleanest and most efficient. 

Conical glasses for receiving specimens of urine. 



APPARATUS 445 

Cover-glasses, one inch square, No. 1 thickness . 
Cover-glass forceps, Stewart's type. 
Dropping bottles, convenient for stains and indicators. 
Erlenmeyer flasks, 100, 250, and 500 cc. capacity. 
Esbach albuminometer. 

Evaporating dish, smooth inner surface, white porcelain, about 
10 cm. diameter. 

Filter papers, in packs, about 12 cm. diameter. 
Funnels, glass, about 5 and 12 cm. diameter. 

Glass tubing, about 2 kg. in assorted sizes. Convenient for making 
pipettes, etc. 

Graduates, 10, 25, and 100 cc. capacity. 

Hemocytometer. The best form has been described in the text. 
Hemoglobinometer. Preferably Sahli's though the other forms 
described in the text may be used. 

Labels, for object slides and for bottles. 
Lens paper. 
Mechanical stage. 
Medicine droppers. 

Microscope. If a Bausch and Lomb be purchased, suitable combina- 
tion of lens includes 5 and 10 oculars, 4 mm. and 16 mm. dry objectives, 
and 1.9 mm. oil-immersion objective mounted on a revolving nose- 
piece. It should be fitted with an Abbe substage condenser, and iris 
diaphragms for stage and condenser. 

Mortar, porcelain, about 8 cm. diameter, and pestle. 
Object slides, 1 by 3 inches, or good quality glass, preferably with 
polished edges. 
Object slide box. 

Pencil, for writing on glass, Blaisdell's No. 163, China marking. 
Petri dishes with covers. 

Pipettes, 5 and 10 cc. capacity, one mark only; 1 cc. capacity, 
graduated in 0.1 cc. 

Ring stand for boiling beakers, etc. 
Stirring rod, glass. 

Stomach tube, either Ewald or Rehfuss. Latter is required if duo- 
denal contents are to be obtained. Pollitzer bulb to fit stomach tube. 
Glass syringe to fit Rehfuss tube. 
Test-tubes, glass, about 16 by 150 mm., with flange. 
Test-tube clamp, wooden. 

Test-tube rack, wooden, with 6 pegs and 6 holes. 
LTrinometer. 

Apparatus required for blood chemistry and quantitative determinations 
on urine (Fig. 54) : , 

Aerating apparatus.— This may be improvised. (See Fig. 55.) 
Analytical balances. 

Burette, 50 cc. capacity, graduated in 0.1 cc. 
Beakers, 100, 250, 500, 1000 cc. capacity. 



446 APPENDIX 

Centrifuge, preferably a heavy one which will take 50 cc. centrifuge 
tubes (Fig. 57), although a smaller centrifuge may be used. 

Centrifuge tubes, 50 cc, round bottom, with pour-out. 

Centrifuge tubes, 50 cc, graduated, with pour-out. 

Centrifuge tubes, 15 cc, graduated, if small centrifuge be used. 

Colorimeter. 

Filter papers, fat-free, about 6 cm. diameter. 

Flasks; Erlenmeyer pattern, 100, 250, 500 cc. capacity; Florence 
pattern, 500 cc, 1 and 2 liter capacity. 

Folin absorption tubes. Folin absorption tube may be made from 
a piece of glass tubing. One end is drawn out to a fine point as though 
a capillary pipette were to be made. This is broken off and sealed in 
the flame and then softened thoroughly by heating. Air is blown into 
the closed tube from the other end until the heated end bulges to form 
a small bulb. A platinum or nichrome wire (mounted in a handle) is 
heated red hot. One side of the glass bulb is heated in the edge of the 
flame to soften an area through which the hot wire is plunged. Three or 
four holes are made in this way. 

Folin's blood sugar tubes (Fig. 54, m). 

Glass beakers, 250 and 500 cc capacity. 

Glass funnels, about 5 cm. and about 15 cm. diameter. 

Glass stirring rods. 

Graduates, cylindrical, accurately graduated, of 10, 25, 50, and 100 
cc capacity. 

Ignition tubes of Pyrex glass, 20 to 25 mm. by 200 mm. Folin's 
tubes are of advantage (Fig. 54, k) . They are graduated at 35 and 50 
cc. They may be obtained from the Emil Greiner Co. 

Micro burner. 

Myers' blood sugar tubes (Fig. 54, I). 

Pipettes.— (a) Ordinary Mohr pipettes with one mark only, of 1, 2, 
3, 4, 5, 10, 20, and 50 cc. capacity (Fig. 54, b). 

(b) Ostwald pipettes, of 1, 2, 3, 5, and 10 cc. capacity 

(Fig. 54, d). 

(c) Folin pipettes (Fig. 54, a). 

(d) Mohr pipettes, graduated, capacity 1 and 2 cc, 

graduated in one-hundredths cc. (Fig. 54, e). 

Quartz pebbles. 

Test-tube rack. 

Tripod and clamp to hold test-tube over burner. 

Volumetric flasks, 10, 20, 25, 50, 60, 100, 500, 1000 cc. capacity 
(Fig. 54, j). 

For quantitative determinations on the urine, the same apparatus will 
be sufficient with the following additions: 

Kjeldahl flasks. 

Kjeldahl distilling apparatus. When many determinations are to be 
made at one time, a special apparatus (Fig. 72) should be obtained. If 
only an occasional specimen be examined, a distilling apparatus may 
be improvised on the principle of a Liebig condenser (Fig. 71). 



APPARATUS 447 

Apparatus Required for Serology (Fig. 32) : 

Animal cages are convenient but wooden boxes may be employed. 

Apparatus for taking blood, MacRae's or the author's (Fig. 32, d 
and e). 

Balances, platform, for balancing loaded tubes before placing in 
centrifuge. 

Centrifuge. While a small, rapid electric centrifuge may be used, a 
heavy centrifuge fitted with two interchangeable heads for 15 and 50 cc. 
tubes, is desirable (Fig. 57) . 

Centrifuge tubes, graduates 15 cc. tubes with conical tip, and 50 cc. 
tubes with round bottom. 

Erlenmeyer flasks, 250, 500 and 1000 cc. capacity. 

Files, triangular. 

Funnels, glass, diameter, 10 to 15 cm. 

Graduates, 100 and 200 cc. capacity. Those with ground glass 
stoppers are convenient for diluting and storing reagents during day's 
work (Fig. 32, c). 

Knives, long amputating or autopsy knife, and small scalpel. 

Pipettes boxes, cylindrical, of copper. 

Pipettes, capillary. These are easily prepared from 6 inch pieces of 
clean, dry glass tubing, which may be held over a Bunsen flame until 
thoroughly softened, and then removed and pulled out until a capillary 
portion of the desired size is obtained. This is allowed to cool, when it 
may be broken to form two pipettes. 

Pipettes, volumetric (Fig. 32, a and b). 
1 cc. capacity, graduated in 0.1 cc. 
1 cc. capacity, graduates in 0.01 cc. 
5 cc. capacity, graduated in 0.05 cc. 
10 cc. capacity, graduated in 0.1 cc. 

Refrigerator. 

Syringes, all glass, Luer type, 2 cc. and 5 cc. capacity. 

Sterilizer, dry air, Lautenschlager type. 

Still, copper with worm condenser, or Barnstead. 

Test-tubes. For the technic described here, employing a bulk of 
2.5 cc. in the completed reaction, a tube about f by 4 inches should be 
used. Tubes of unusually resistant glass should be used. These may 
be obtained from the Steele Glass Co., Locust St., Philadelphia. 

Test-tube racks, of metal, to fit water-bath (Fig. 35) . 

Water-bath or incubator, maintained at temperature of 37° C. If 
only a small equipment is purchased, the bacteriological dry air incu- 
bator may be used, but longer incubation will have to be employed. 
The water-bath should be deep enough to permit immersion of the 
tubes to one-half their depth, and is heated preferably with electricity, 
automatically controlled (Fig. 36). 

Water-bath maintained at a temperature of 56° C. A simple water- 
bath may be used, or it may be fitted with a thermoregulatory device. 

Wire baskets for holding test-tubes, 



448 APPENDIX 



CARE OF GLASSWARE FOR SEROLOGICAL WORK. 

It is absolutely essential that all glassware be chemically clean. 
Bacteriological sterility is desirable but not absolutely necessary except 
in securing and handling blood which is to be preserved as amboceptor, 
or specimens of human blood which are to be held some days before 
examination. 

Test-tubes employed in the reaction should be emptied immediately 
after use. If the process of cleaning can be finished at once, a test- 
tube brush should be used to remove any sediment of blood, the tubes 
rinsed with several changes of tap water, then with distilled water and 
finally placed upside down in wire baskets and put in dry-air steril- 
izer for an hour at 200° C. When cleaning cannot be finished at once, 
the contents of the tubes should be poured out and the tubes then put 
to soak in clear water until the foregoing procedure can be carried out. 
Blood clots should be shaken out as soon as the reactions are completed, 
the tube brushed vigorously a number of times with a test-tube brush, 
rinsed with several changes of clear water and then placed in clear 
water for several hours when the cleaning may be completed as pre- 
viously outlined. Flasks and graduated cylinders may be cleaned with 
repeated changes of water and rinsed with distilled water. Brushing 
is rarely necessary. Pipettes should be given scrupulous care. Im- 
mediately after use they should be placed in tall cylinders filled with 
distilled water in order to prevent drying and coagulation of the serum. 
Later the cotton plugs may be extracted with a barbed dental broach 
(Fig. 32, h). Then the upper end of each pipette in turn should be 
inserted into the rubber-tubing outlet of a reservoir bottle of distilled 
water and the water should be allowed to flow through. It is advisable 
to soak the pipettes after this in a flat enamel basin with distilled water, 
and to repeat the process or running distilled water through in twenty- 
four hours. When thoroughly clean the pipettes may be dried in the 
oven and the upper ends then lightly packed with a little cotton 
(Fig. 32, a and b). Capillary pipettes are discarded after using once. 
It is a waste of time to attempt to clean them properly. 

Glassware should always be transparent, bright, shiny, and free 
from the evidence of dried serum or blood. This is easy to accomplish 
if the simple methods of cleaning just outlined are employed. The 
routine use of chemical cleaning fluids is both unnecessary and unwise 
on account of the possibility of insufficient rinsing. Dependence should 
be placed upon brushing, wiping, and the copious use of water. When 
tubes become smoky and dried serum occludes the pipettes, the glass- 
ware may be boiled for thirty minutes in 2 to 5 per cent, sodium 
carbonate, rinsed in clear water until litmus paper fails to show any 
trace of alkali, and finally cleaned by the use of distilled water and dried 
in the hot air sterilizer. 



PREPARATION OF AUTOGENOUS VACCINES 449 



PREPARATION OF AUTOGENOUS VACCINES. 

Preparation of the Emulsion.— The organisms are isolated and obtained 
in pure culture in the usual way. This should be accomplished as 
rapidly as possible. When a pure culture is obtained, several tubes 
of solid medium should be planted, selecting a type of medium most 
suitable for the growth of the organism. The cultures are allowed to 
grow for twenty-four hours at 37° C, when the tubes are filled with 
sufficient sterile normal salt solution to entirely submerse the slanted 
medium. A platinum wire, mounted on a glass rod, is bent into a wide 
loop. This is sterilized in the Bunsen flame. After it has cooled, it is 
used to scrape the bacterial growth off the slanted medium and to mix 
it with the salt solution. The bacterial emulsion thus prepared is 
poured from all the tubes into a dry sterile test-tube or bottle, con- 
taining a few bits of broken glass. The ends of the tubes should be 
passed through the flame just before the emulsion is poured out, in 
order to avoid contaminating the emulsion. If a. test-tube is used, it 
should be hermetically sealed with a blow pipe, while a bottle may be 
stoppered with a sterile rubber stopper or tight-fitting glass stopper. 
The emulsion is then shaken vigorously for fifteen to twenty minutes. 
A shaking machine expedites this part of the preparatory labor. The 
vaccine is now ready for counting. 

Standardization According to a Modified Wright's Method.— The worker 
should provide himself with a capillary pipette the capillary portion 
of which should be at least six inches long and of fairly uniform bore. 
A mark is made at the capillary portion of the pipette about 1 cm. 
from the end with ink or a grease pencil and the larger end of the pipette 
is fitted with a rubber nipple. Such a pipette may be readily drawn 
from a piece of glass tubing. 

The vaccine should be thoroughly shaken before counting. Two or 
three drops of well-shaken emulsion are placed in a watch-crystal . The 
worker then pricks his finger and allows a drop of blood to fall on a slide. 
This blood is drawn up into the pipette to the mark. After having thus 
measured off a quantity of blood, the column of blood is aspirated 
carefully up the pipette so that its base will occupy a position about 
5 mm. above the tip of the pipette, and the bacterial emulsion is drawn 
up to the mark. In the pipette there now should be a column of blood 
and a column of bacterial emulsion of equal height separated by an air 
space about 5 mm. long. The contents of the pipette are carefully 
ejected onto a clean slide and are then drawn back and forth into and 
out of the pipette three or four times to mix the blood and the organisms 
thoroughly. It is desirable to avoid the admixture of air bubbles. The 
drop is then spread on the slide in the manner described for making 
blood smears, stained with Wright's stain, and counted. In a 
number of different fields, selected at random, the number of red blood 
cells and organisms are enumerated to determine their ratio. At least 
1000 red cells should be counted. The preparation should be discarded 
29 



450 APPENDIX 

and another made if the bacteria prove to be unevenly distributed. In 
making the count, an Ehrlich's ocular is of the utmost convenience, 
since the visible field may be greatly reduced, making the operation of 
counting much less trying. If this instrument is not available, a sub- 
stitute may be made by cutting a circular piece of paper to rest on the 
platform which is found between the upper and lower lenses of all 
oculars, and by making a small hole in center. 

Calculation.— Assume that the worker's blood be normal, containing 
five million red blood corpuscles per cm. and that equal quantities of 
blood and bacteria suspension were taken. The number per cm. is 
computed by use of this equation, in which A = the number of bacteria 
counted, B = the number of corpuscles counted, and X represents the 
number of organisms per cm.:— 5.000,000 X A 

_ =X 

B 

Standardization According to Hopkins' Method.— The writer much 
prefers Hopkins' method. It requires a rapid centrifuge and an 
specially graduated centrifuge tube (Hopkins' tube) 1 (Fig. 136). 

The bacterial emulsion is prepared as directed in the preceding 
method and is poured into the Hopkin's tube which is centrifugalized 
for thirty minutes at a rate of 2800 revolutions per second in a centri- 
fuge whose head has a diameter of 18 cm. from the tip of one tube to 
the tip of the other. Then the supernatant salt is removed carefully 
with bacteria to reduce the top of the sediment to the mark desired. 
If the mark be .05, fresh salt solution may be added to the mark 1.0 
and a 0.5 per cent, dilution is obtained. If the bacterial sediment 
comes up to the mark 0.1 and salt solution is added to the mark 5, 
we have a 2 per cent, dilution. Hopkins has prepared a table showing 
the number of bacteria represented in a 1 per cent, dilution by the 
different forms. 

Billion 
Per cent. percc. 

Staphylococcus aureus and albus 1 10 

Streptococcus haemolyticus 1 8 

Gonococcus 1 8 

Pneumococcus 1 2 

Bacillus typhosus 1 8 

Bacillus coli 1 4 

When the salt has been added to make an emulsion of the desired 
strength, the vaccine is poured in a sterile bottle or test-tube and is 
ready for sterilization. 

Sterilization of the Vaccine.— The bottle or tube containing the vac- 
cine is placed in a water-bath, and the water is kept at a temperature 
of 65° C. for one hour. At the end of that time, cultures are made 
from the vaccine on two or more tubes of the same sort of medium as 

1 Those may be obtained from (lie International Equipment Co., Cambridge, Mass. 



WEIGHTS AND MEASURES 451 

was employed for growing the cultures. These cultures are allowed 
to incubate at least twenty-four hours. If sterile, the vaccine may be 
employed. 

Dilution and Preservation of Vaccines.— If the sterilized suspension 
be stronger than desired for administration, the vaccine should be 
diluted with sterile salt solution so that 0.1 cc. of the dilution contain 
the initial dose. Suppose, for example, that the sterilized and stand- 
ardized emulsion is found to contain 500,000,000 bacteria per cc, and 
that the desired initial dose, which we wish to have in bulk of 0.1 cc, 
is 50,000,000, we would take 1 cc. of the emulsion and add 9 cc. of 
sterile salt solution. 

Lysol or tricresol is added as a preservative, 3 drops being added 
with a sterile dropper to each 10 cc. of completed vaccine. 

WEIGHTS AND MEASURES. 
METRIC SYSTEM. 



Meter (unit of length) : 






Millimeter (mm.) 


= 


1/1000 meter 


Centimeter (cm.) 


= 


1/100 


Kilometer 


= 


1000 


Micron 


= 


1/1000 millimeter 


Gram (unit of weight) (gm.) : 






Milligram (mg.) 


= 


1/1000 gram 


Kilogram (kg.) 


= 


1000 " 


Liter (unit of capacity) (L.) : 






Cubic centimeter 


= 


1/1000 liter 



[same as milliliter (ml)] 

APOTHECARIES' OR TROY WEIGHT. 

(Used in Prescriptions) 

Grain (gr.) : 

[Scruple O) = 20grs.] 

Dram (5) [ = 33] = 60 grs. 

Troy ounce (§) = 85 = 480 grs. 
[Troy pound. = 12g = 5760 grs.] 

(Bj of water under standard conditions measures 504.83 minims.) 

AVOIRDUPOIS WEIGHT. 

(A System Used in Commerce) 

Grain = same as Troy grain 

Ounce (oz.) = 437j^ grains 

Pound (lb.) = 16 ozs. = 7000 grains 

Ton = 2000 lbs. 

UNITED STATES APOTHECARIES' OR WINE MEASURE. 
(Used in United States for Both Prescription and Commercial Purposes) 

Minim (Til) (approximately equal to 1 drop or to 1 grain of water — more exactly, 
0.95 grain) 

Fluidrachm (fl3) = 60 m 

Fluidounce (A3) = 8 A3 = 480111 (AS j of water under 

standard conditions weighs 450?- grains). 
Pint (pt„ or Octarius, O) = 16 flg = 7680 111 

Quart (qt.) = 2 pts. = 32 fig 

Gallon (gal., or Congius, C) = 8 = 128 fig = 61,440 111 
A gallon Isolds 231 cubic inches. 



45: 



APPENDIX 



EQUIVALENTS OF METRIC AND COMMON SYSTEMS. 
Space. 



1 meter = 39.370 inches 




1 inch 


= 0.0254 M. = 2.54 cm, 




= 3. ft. 3.370 inches 


1 ft. 


= 30.227 cm. 




= 1 yd. 3.370 inches 


1yd. 


= 91.440 cm. 






Capacity (United States) 




1 cc. 


= 16.23 1U 




11 


= 0.06161 cc. 


11. 


= 33.815 A3 




lflS 


= 3.7 cc. 




= 2.113 pts. 




1A5 


= 29.574 cc. 




= 0.2642 gal. 




1 pt. 
lgal. 

Capacity (British) 


= 0.4731 1. 
= 3.7854 1. 


1 I. 


= 1.760 pints 




1 pint 


= 0.56791. 




= 0.2209 gallons 




1 gallon 

Weight. 


= 4.5435 1. 


1 mg. 


= eV gr. 




lgr. 


= 64.8 mg. = 0.0648 gm. 


1 gm. 


= 15.432 grs. 




1 5 


= 4 gm. 




= 0.03527 oz. Av. 


1 oz. Av. 


= 28.3495 gm. 




= 0.03215 3 Troy 


1 3 Troy 


= 31.1035 gm. 


1kg. 


= 2.2046 lbs. 




1 lb. 


= 0.4536 kg. 






Approximate Equivalents. 




1 gm. 


1 = 15.5 grs. or minims 1 gr. or minim 


= 065 gm. or cc. 


1 cc. 


/ 




1 dram 


= 4.0 cc. (— ) 


1 mg. 


= £% grain 




1 ounce 


= 30.0 cc. (— ) 


1 liter 


= 1 quart ( +) 




1 pint 


= 0.5 liter (— ) 


1kg. 


= 2.2 lbs. 









WEIGHTS AND MEASURES 



453 



OONVERSION OF CENTRIGRADE TO FAHRENHEIT DEGREES 



°c. 


°F. 


°C. 


°F. 


°C. 


°F. 


°C. 


°F. 


°C. 


°F. 


-30 


-22.0 


+27 


+80.6 


+83 


+ 181.4 


+ 139 


+282.2 


+ 195 


+383.0 


-29 


-20.2 


28 


82.4 


84 


183.2 


140 


284.0 


196 


384.8 


-28 


-18.4 


29 


84.2 


85 


185.0 


141 


285.8 


197 


386.6 


-27 


-16.6 


30 


86.0 


86 


186.8 


142 


287.6 


198 


388.4 


-26 


-14.8 


31 


87.8 


87 


188.6 


143 


289.4 


199 


390.2 


-25 


-13.0 


32 


89.6 


88 


190.4 


144 


291.2 


200 


392.0 


-24 


-11.2 


33 


91.4 


89 


192.2 


145 


293.0 


201 


393.8 


-23 


-9.4 


34 


93.2 


90 


194.0 


146 


294.8 


202 


395.6 


-22 


-7.6 


35 


9..0 


91 


195.8 


147 


296.6 


203 


397.4 


-21 


-5.8 


36 


96.8 


92 


197.6 


148 


298.4 


204 


399.2 


-20 


-4.0 


37 


98.6 


93 


199.4 


149 


300.2 


205 


401.0 


-19 


-2.2 


38 


100.4 


94 


201.2 


150 


302.0 


206 


402.8 


-18 


0.4 


39 


102.2 


95 


203.0 


151 


303.8 


207 


404.6 


-17 


+1.4 


40 


104.0 


96 


204.8 


152 


305.6 


208 


406.4 


-16 


3.2 


41 


105.8 


97 


206.6 


153 


307.4 


209 


408.2 


-15 


5.0 


42 


107.6 


98 


208.4 


154 


309.2 


210 


410.0 


-14 


6.8 


43 


109.4 


99 


210.2 


155 


311.0 


211 


411.8 


-13 


8.6 


44' 


111.2 


100 


212.0 


156 


312.8 


212 


413.6 


-12 


10.4 


45 


113.0 


101 


213.8 


157 


314.6 


213 


415.4 


-11 


12.2 


46 


114.8 


102 


215.6 


158 


316.4 


214 


417.2 


-10 


14.0 


47 


116.6 


103 


217.4 


159 


318.2 


215 


419.0 


-9 


15.8 


48 


118.4 


104 


219.2 


160 


320.0 


216 


420.8 


-8 


17.6 


49 


120.2 


105 


221.0 


161 


321.8 


217 


422.6 


-7 


19.4 


50 


122.0 


106 


222.8 


162 


323.6 


218 


424.4 


-6 


21.2 


51 


123.8 


107 


224.6 


163 


325.4 


219 


426.2 


-5 


23.0 


52 


125.6 


108 


226.4 


164 


327.2 


220 


428.0 


-4 


24.8 


53 


127.4 


109 


228.2 


165 


329.0 


221 


429.8 


-3 


26.6 


54 


129.2 


110 


230.0 


166 


330.8 


222 


431.6 


-2 


28.4 


55 


131.0 


111 


231.8 


167 


332.6 


223 


433.4 


-1 


30.2 


56 


132.8 


112 


233.6 


168 


334.4 


224 


435.2 





32.0 


57 


134.6 


113 


235.4 


169 


336.2 


225 


437.0 


+ 1 


33.8 


58 


136.4 


114 


237.2 


170 


338.0 


226 


438.8 


2 


35.6 


59 


138.2 


115 


239.0 


171 


339.8 


227 


440.6 


3 


37.4 


60 


140.0 


116 


240.8 


172 


341.6 


228 


442.4 


4 


39.2 


61 


141.8 


117 


242.6 


173 


343.4 


229 


444.2 


5 


41.0 


62 


143.6 


118 


244.4 


174 


345.2 


230 


446.0 


6 


42.8 


63 


145.4 


119 


246.2 


175 


347.0 


231 


447.8 


7 


44.6 


64 


147.2 


120 


248.0 


176 


348.8 


232 


449.6 


8 


46.4 


65 


149.0 


121 


249.8 


177 


350.6 


233 


451.4 


9 


48.2 


66 


150.8 


122 


251.6 


178 


352.4 


234 


453.2 


10 


50.0 


67 


152.6 


123 


253.4 


179 


354.2 


235 


455.0 


11 


51.8 


68 


154.4 


124 


255.2 


180 


356.0 


236 


456.8 


12 


53.6 


69 


156.2 


125 


257.0 


181 


357.8 


237 


458.6 


13 


55.4 


70 


158.0 


126 


258.8 


182 


359.6 


238 


460.4 


14 


57.2 


71 


159.8 


127 


260.6 


183 


361.4 


239 


462.2 


15 


59.0 


72 


161.6 


128 


262.4 


184 


363.2 


240 


464.0 


16 


60.8 


73 


163.4 


129 


264.2 


185 


365.0 


241 


465.8 


17 


62.6 


74 


165.2 


130 


266.0 


186 


366.8 


242 


467.6 


18 


64.4 


75 


167.0 


131 


267.8 


187 


368.6 


243 


469.4 


19 


66.2 


76 


168.8 


132 


269.6 


188 


370.4 


244 


471.2 


20 


68.0 


77 


170.6 


133 


271.4 


189 


372.2 


245 


473.0 


21 


69.8 


78 


172.4 


134 


273.2 


190 


374.0 


246 


474.8 


22 


71.6 


79 


174.2 


135 


275.0 


191 


375.8 


247 


476.6 


23 


73.4 


80 


176.0 


136 


276.8 


192 


377.6 


248 


478.4 


24 


75.2 


81 


177.8 


137 


278.6 


193 


379.4 


249 


480.2 


25 


77.0 


82 


179.6 


138 


280.4 


194 


381.2 


250 


482.0 


26 


78.8 



















To convert degrees Centigrade to degrees Fahrenheit, multiply by | and add 32. 

To convert degrees Fahrenheit to degrees Centigrade, subtract 32 and multiply by |- 



454 BIBLIOGRAPHY 

BIBLIOGRAPHY. 

TEXT-BOOKS AND MONOGRAPHS. 

American Public Health Association: Standard Methods for the Examination of 

Water and Sewage. 4th edition, 1920, p. 94. 
Arneth, Prof.: Die Neutrophilen weissen Blutkorperchen bei Infections-Krankheiten. 

Fischer, Jena, 1904. 
Avery, O. T., Chickering, H. T., Cole, R., and Dochez, A. R.: Acute Lobar Pneu- 
monia. Prevention and Serum Treatment. Monograph No. 7. Rockefeller 

Institute for Medical Research, 1917. New York. 
Barker, L. F.: Monographic Medicine. Appleton & Co., New York and London, 1916. 
Bass, C. C, and Johns, F. M.: Alveolodental Pyorrhea. W. B. Saunders Co., Phila- 
delphia, 1915. 
Boothby, W. M., and Sandiford, Irene: Laboratory Manual of the Technic of Basal 

Metabolic Rate Determination. W. B. Saunders Co., Philadelphia, 1920. 
Brown, J. H.: The Use of Blood Agar for the Study of Streptococci. Monograph 

No. 9, Rockefeller Institute for Medical Research, 1919, New York. 
Cabot, R. C: Aplastic Anemia. Osier's Modern Medicine, 4, 637. Lea & Febiger, 

Philadelphia, 1908. 
Cabot, Richard C: Clinical Examination of the Blood. Fifth edition. Wm. Wood 

& Co , New York, 1904. 
Cabot, R. C: Leukemia. Osier's Modern Medicine, 4, 668. Lea & Febiger, Phila- 
delphia, 1908. 
Chamot, E. M.: Elementary Chemical Microscopy. John Wiley & Sons, Inc., New 

York, 1916. 
Christian, Henry A.: Purpura and Allied Conditions. Oxford Medicine, 2, 779. 
Craig, C. F.: The Wassermann Test. Mosby Co., St. Louis, 1918. 
Craig, C. F.: The Malarial Fevers. Wood & Co., New York, 1919. 
Craig, F. A.: A Study of the Blood in Pulmonary Tuberculosis. Fourth Annual 

Report, Henry Phipps Institute for the Study, Treatment, and Prevention of 

Tuberculosis. 1909, p. 108. 
Dock, G., and Bass, C. C: Hookworm Disease, p. 178. Mosby Co., 1910. 
Drinker, Cecil K.: Diseases of the Blood. The Pathological Physiology of Blood 

Cell Formation and Blood Cell Destruction. Oxford Medicine, 2, 509. 
Ehrlich, P.: Anemia: Histology of the Blood, Normal and Pathologic. Nothnagel's 

Practice of Medicine. W. B. Saunders Co., 1905. 
Emerson, C. P.: Clinical Diagnosis. Lippincott Co., Philadelphia. 
Ewing, James: Clinical Pathology of the Blood. Lea Brothers & Co., Philadelphia, 

1901. 
Fantham, H. B., Stephens, J. W. W., and Theobold, F. V.: The Animal Parasites of 

Man. Wood & Co., New York, 1916. 
Farrington and Woll: Testing Milk and its Products. 24th edition. Mendota Book 

Co., 1918. 
Fitz, R.: Polycythemia. Oxford Medicine, 2, 763. 
Folin, Otto: Determination of Acidity of Urine. Laboratory Manual of Biological 

Chemistry, p. 127, 2d edition. Appleton & Co., New York, 1919. 
Gay, F. P.: Typhoid Fever: Considered as a Problem of Scientific Medicine. The 

Macmillan Co., New York, 1918. 
Gruner, O. C: The Biology of the Blood Cells. Wm. Wood & Co., New York, 1914. 
Gulland, G. Lovell, and Goodall, Alexander: The Blood. Second edition. E. B. 

Treat & Co., New York, 1915. 
Hand-book of Chemistry and Physics. Chemical Rubber Co., Cleveland, 1919. 
Hawk, P. B.: Practical Physiological Chemistry. P. Blakiston's Son & Co., Phila- 
delphia. 
Hayem, G.: Du Sang et de ses Alterations Anatomiques. Masson et Cie, Paris, 1889. 
Hensel, O., Weil, R., and Jelliffe, S. E.: The Urine and Feces in Diagnosis, p. 271. 

Lea Brothers & Co., Philadelphia, 1905. 
Hiss, P. H., and Zinsser, H.: A Text-book of Bacteriology. Appleton & Co., New 

York, 1916. 
Hoffman, Prof., and Schmidt, P.: (Quoted by Oliver, Lead Poisoning, p. 125). Hoeber, 

New York, 1914. 
Karsner, H. T., and Ecker, E. E.: Principles of Immunology. J. B. Lippincott Co., 

Philadelphia, 1921. 



TEXT-BOOKS AND MONOGRAPHS 455 

Kendall, A. I.: Bacteriology, General, Pathological and Intestinal. Lea & Pebiger, 

Philadelphia, 1921. 
Kolfner, J. A.: Infection, Immunity and Specific Therapy. 2d edition. W. B. 

Saunders Co., Philadelphia, 1917. 
Levinson, A.: Cerebrospinal Fluid in Health and Disease. Mosby Co., St. Louis, 1919. 
McLean, Jay: Hemophilia. Oxford Medicine, 2, 799. 

MacCallum, W. G.: Text-book of Pathology, p. 761. W. B. Saunders Co., Philadel- 
phia, 1916. 
MacLeod, J. J. R. : Physiology and Biochemistry in Modern Medicine, p. 519. Mosby 

Co., St. Louis, 1918. 
Mallory, F. B., and Wright, J. H.: Pathological Technique. W. B. Saunders Co., 

Philadelphia, 1918. 
Minot, George R. : Diseases of the Blood. Clinical Discussion of the Anemias. Oxford 

Medicine, 2, 589. 
Myers, Victor C: Practical Chemical Analysis of Blood. C. V. Mosby Co., St. Louis, 

1921. 
Noguchi, H. : Serum Diagnosis of Syphilis and the Butyric Acid Test for Syphilis. 

2d edition. J. B. Lippincott Co., Philadelphia, 1911. 
Nuttall, G. H. F., and Graham-Smith, G. S.: The Bacteriology of Diphtheria. Uni- 
versity Press, Cambridge, 1913. 
Ordway, T. O., and Gorham, L. W.: Leukemia. Oxford Medicine, 2, 681. 
Oxford Medicine, Volume II. Oxford University Press. (Especially the following 
articles: The Pathological Physiology of Blood-cell Formation and Blood-cell 
Destruction, by Cecil K. Drinker; Clinical Discussion of the Anemias, by George 
R. Minot; Leukemia, by Thomas Ordway and L. Whittington Gorham; Poly- 
cythemia, by Reginald Fitz; Purpura and Allied Conditions, by Henry A. Christian; 
Hemophilia, by Jay McLean.) 
Park, W. H., and Williams, A.: Pathogenic Microorganisms. Lea & Febiger, Phila- 
delphia, 1920. 
Pearce, H. M., Krumbh aar, E. B., and Frazier, C. H. : The Spleen and Anemia Experi- 
mental and Clinical Studies. J. B. Lippincott Co., Philadelphia, 1918. 
Pottenger, F. M.: Clinical Tuberculosis, 1, 560. Mosby Co., St. Louis, 1917. 
Rieder, Herman, and Delepine, A. S. : Atlas of Urinary Sediments. Charles Griffin & 

Co., London, 1899. 
Schleip, Karl: Hematological Atlas. The Rebman Co., New York, 1920. 
Schmidt, Ad., and Strasburger, J.: Die Faeces des Menschen. A. Hirschwald, Berlin, 

1905. 
Sellards, A. W. : The Principles of Acidosis and Clinical Methods for its Study. Har- 
vard Univ. Press, Cambridge, 1919. 
Simon, C. E.: A Manual of Clinical Diagnosis by Means of Laboratory Methods. Lea 

& Febiger, Philadelphia, 1914. 
Simon, C. E.: Human Infection Carriers: Their Significance, Recognition and 

Management. Lea & Febiger, Philadelphia, 1919. 
Stitt, E. R.: Practical Bacteriology, Blood Work and Animal Parasitology. P. 

Blakiston's Son & Co., Philadelphia, 1916. 
Sutton, W. L.: Volumetric Analysis. 9th edition. P. Blakiston's Son & Co., Phila- 
delphia, 1904. 
Todd: Clinical Diagnosis. Fourth edition. W. B. Saunders Co., Philadelphia, 1918. 
Tyson, James: The Practice of Medicine. Fourth edition. P. Blakiston's Son & 

Co. Philadelphia, 1906. (Especially the section on Animal Parasites.) 
Von Jaksch, Rudolph: Clinical Diagnosis. Fifth English edition. Edited by A. E. 

Garrod. Charles Griffin & Co., London, 1905. 
War Manual No. 6: Laboratory Methods of the United States Army, p. 119. Lea 

& Febiger, Philadelphia, 1918. 
Ward, G. R.: Bedside Hematology. W. B. Saunders Co., Philadelphia, 1914. 
Webster, R. W. : Diagnostic Methods, Chemical, Bacteriological and Microscopical. 

P. Blakiston's Son & Co., Philadelphia, 1920. 
Wood, F. C: Chemical and Microscopical Diagnosis. Appleton & Co., New York, 1917. 
Wood, F. C, Vogel, K. M., and Famulener, L. W.: Laboratory Technic. Daugherty, 

New York, 1917. 
Zinsser, H.: Infection and Resistance, p. 208. The Macmillan Co., New York, 1914. 
Zinsser, Hans, Hopkins, J. K., and Ottenburg, R.: Laboratory Course in Serum Study. 
The Macmillan Co., New York, 1916. 



456 BIBLIOGRAPHY 



ORIGINAL ARTICLES 

Appleton, V. B.: Determination of Hemoglobin during Infancy by the Palmer and 

Van Slyke Methods. Jour. Biol. Chem., 1918, 24, 369. 
Ashby, W. : The Determination of the Length of Life of Transfused Blood Corpuscles 

in Man. Jour. Exp. Med., 1919, 29, 267. 
Austin, J. H., and Van Slyke, D. D.: Determination of Chlorides in Whole Blood. 

Jour. Biol. Chem., 1920, 41, 345. 
Avery, O. T. : A Selective Medium for B. influenzae. Oleate-hemoglobin Agar. Jour. 

Am. Med. Assn., 1918, 71, 2050. 
Avery, O. T. : Determination of Types of Pneumococcus in Lobar Pneumonia. Jour. 

Am. Med. Assn., 1918, 70, 17. 
Barker, L. F.: On the Presence of Iron in the Granules of the Eosinophile-leukocytes, 

Johns Hopkins Hosp. Bull., 1894, 5, 93. 
Bass, C. C, and Johns, F. M.: The Cultivation of Malarial Plasmodia (Plasmodium 
Vivax and Plasmodium Falciparum) in Vitro. Jour. Exper. Med., 1912, 16, 567. 
Bass, C. G, and Johns, F. M.: A Method of Concentrating Malarial Plasmodia for 

Diagnostic and Other Purposes. Am. Jour. Trop. Diseases, 1915, 3, 298. 
Benedict, A. L. : Indicators in Gastric Analysis, with Special Reference to Tropeolin 00. 

Med. News, 1904,84, 597. 
Benedict, A. L.: Correspondence. Cleveland Med. Jour., 1918, 17, 279. 
Benedict, S. R. : Studies in Creatine and Creatinine Metabolism. I. The Preparation 

of Creatine and Creatinine from Urine. Jour. Biol. Chem., 1914, 18, 184. 
Benedict, S. R., and Hitchcock, E. H.: On the Colorimetric Estimation of Uric Acid in 

Urine. Jour. Biol. Chem., 1915, 20, 619. 
Blankenhorn, M. A.: The Bile Content of the Blood in Pernicious Anemia. Arch. 

Int. Med., 1917, 19, 344. 
Blankenhorn, M. A.: The Distribution of Bile in Certain Types of Jaundice. Arch. 

Int. Med., 1918, 21, 282. 
Boas, H.: Die Wasserrnann'sche Reaktion mit bes. Beriicksichtigung ihrer klinischen 
Verwertbarkeit. (Quoted in Special Report Series, No. 21, Med. Research Com. 
London, 1918, p. 6). 
Bock, J. C, and Benedict, S. R.: A New Form of Colorimeter. Jour. Biol. Chem., 

1918, 35, 227. 
Boggs, T. R., and Pincoffs, M. C: A Case of Pulmonary Moniliasis in the United 

States. Johns Hopkins Hosp. Bull., 1915, 26, 407. 
Briggs, L. H. : Clinical Value of the Arneth Method of Blood Examination. California 

State Jour. Med., 1912, 10, 337. 
Brown, L., and Petroff, S. A.: The Clinical Value of Complement-fixation in Pulmon- 
ary Tuberculosis Based on a Study of 540 Cases. Am. Rev. Tuberc, 1918, 2, 253. 
Buckman, T. E., and Hallisey, J. E.: Studies in the Properties of Blood Platelets. A 

New Method for Counting Platelets. Jour. Am. Med. Assn., 1921, 76, 427. 
Bushnell, G. E., and Treuholtz, C. A.: Arneth's Method in the Clinical Study ol 

Pulmonary Tuberculosis. Med. Rec, 1908, 73, 471. 
Cabot, R. C: Ring Bodies (Nuclear Remnants?) in Anemic Blood. Jour. Med. Res. 

1903, 9, 15. 
Capps, J. A.: A Study of Volume Index. Observations upon the Volume of Erythro 

cytes in Various Disease Conditions. Jour. Med. Res., 1903, 10, 367. 
Chace, A. F., and Myers, V. C: The Value of Recent Laboratory Tests in the Diag 
nosis and Treatment of Nephritis, with Special Reference to the Chemical Exami 
nation of the Blood. Jour. Am. Med. Assn., 1916, 67, 929. 
Chace, A. F., and Myers, V. C: Acidosis in Nephritis. Jour. Am. Med. Assn., 1920 : 

74, 641. 
Chatard, J. A.: The Leukocytes in Acute Lobar Pneumonia. Johns Hopkins Hosp 

Rep., 1910, 15, 89. 
Chauffard, A.: (Quoted by Pearce) Pathogenie de l'ictere congenital de l'adulte 

Semaine Med., 1907, 27, 25. 
Clark, W. M.: The "Reaction" of Bacteriologic Culture Media. Jour. Infec. Dis. 

1915, 17, 109. 
Clark, W. M., and Lubs, H. A.: The Colorimetric Determination of Hydrogen-ion 
Concentration and its Applications in Bacteriology. Jour. Bacteriol., 1917, 2, 
1, 109 and 191. 
Cole, S. W., and Onslow, R. : On a Substitute for Peptone and a Standard Nutrient 
Medium for Bacteriological Purposes. Lancet, 1916, 2, 9 and 1011. 



ORIGINAL ARTICLES 457 

Covvdry, E. V.: The Staining of Mitochondria in Human Lymphocytes with Janus 

Green. Anat. Rec, 1914, 8, 140. 
Craig, C. F. : The Relation of Certain Bacteria to Non-specific Reactions with the 

Complement-fixation Test for Lues. Jour. Exp. Med., 1911, 13, 521. 
Craig, C. F.: The Results and Interpretation of the Wassermann Test. Am. Jour. 

Med. Sci., 1915, 149, 41. 
Craig, C. F. : Observations upon Complement-fixation in the Diagnosis of Pulmonary 

Tuberculosis. Am. Jour. Med. Sci., 1915, 150, 782. 
Craig, C. F., and Nichols, H. J.: The Effect of the Ingestion of Alcohol on the Result 

of the Complement-fixation Test in Syphilis. Jour. Am. Med. Assn., 1914, 57, 474. 
Cummer, C. L. : Device for Withdrawing Blood from Veins. Jour. Lab. and Clin. 

Med., 1919-20, 5, 257. 
Daland, J.: A New Hematokrit and a New Technique. Tr. Coll. Phys. Philadelphia. 

1894, 16, 192. 
Dexter, R., and Cummer, C. L. : The Occurrence of Native Antisheep Amboceptor in 

Human Serum and its Importance in the Performance of the Wassermann Reac- 
tion. Arch. Int. Med., 1912, 9, 605. 
Dexter, R., and Cummer, C. L.: The Importance of the Early Diagnosis of Syphilis. 

Jour. Am. Med. Assn., 1912, 59, 1254. 
Dobell, C: On the Nomenclature of the Spirochete of Syphilis. Medical Research 

Committee. Special Report, No. 19, p. 47. London, 1918. 
Dock, G.: Methods, Value and Limitations of the Knowledge of the Gastric Con- 
tents. Jour. Am. Med. Assn., 1905, 45, 1385. 
Dohle, Prof. Dr.: Leukocyteneinschlusse bei Scharlach. Centralbl. f. Bact., 1 Abt. 

Orig.; 1911,61, 63. 
Dreyer, G, and Inman, A. C: The Agglutination Curve and its Importance in the 

Diagnosis of Typhoid and Paratyphoid Fevers in Inoculated Persons. Lancet, 

1817, 1, 363. 
Drinker, C. K., and Hurwitz, S. H. : The Factors of Coagulation in Primary Pernicious 

Anemia. Arch. Int. Med., 1915, 15, 733. 
Duke, W. W. : The Relation of Blood Platelets to Hemorrhagic Disease. Jour. Am. 

Med. Assn., 1910, 55, 1185. 
Ecker, E. E. : An Apparently Practical Method for the Isolation of Pfeiffer's Bacillus 

from Sputum. Jour. Am. Med. Assn., 1918, 71, 1482. 
Ecker, E. E., and Sasano, K.: A Rapid Method for Preparing Antigens from Normal 

Heart Muscle. Jour. Infec. Dis., 1919, 25, 174. 
Einhorn, M., and Rosenbloom, J.: A Study of the Duodenal Contents in Man. Arch. 

Int. Med., 1910, 6, 666. 
Einhorn, M. : A Practical Method of Obtaining the Duodenal Contents in Man. Med. 

Rec. 1910, 77, 98. 
Einhorn, M.: Historical Sketch of the Development of the Duodenal Tube. Am. 

Jour. Med. Sci., 1916, 151, 202. 
Fildes, P., and Mcintosh, J.: The Wassermann Reaction and its Application to Neur- 
ology. Brain, 1913-14, 36, 193. 
Foiin, Otto: Approximately Complete Analysis of Thirty "Normal" Urines. Am. 

Jour. Physiol., 1905, 13, 45. 
Folin, Otto, and Denis, W. : New Methods for the Determination of Total Non-protein 

Nitrogen, Ujrea and Ammonia in Blood. Jour. Biol. Chem., 1912, 11, 527. 
Folin, Otto, and Denis, W. : On the Colorimetric Determination of Uric Acid in Urine. 

Jour. Biol. Chem., 1913, 14, 95. 
Folin, Otto, and Denis, W.: On the Creatinine and Creatine Content of the Blood. 

Jour. Biol. Chem., 1914, 17, 487. 
Folin, Otto, and Denis, W. : Nitrogen Determination by Direct Nesslerization. The 

Use of the Colorimeter. Jour. Biol. Chem., 1916, 26, 484. 
Folin, Otto, and Doisy, E. A.: Impure Picric Acid as a Source of Error in Creatine 

and Creatinine Determination. Jour. Biol. Chem., 1916-17, 28, 349. 
Folin, O., and Farmer, C. J.: A New Method for the Determination of Total Nitrogen 

in Urine. Jour. Biol. Chem., 1912, 11, 493. 
Folin, O., and MacCallum, A, B.: A New Method for the (Colorimetric) Determina- 
tion of Uric Acid in Urine. Jour. Biol. Chem., 1913, 13, 363. 
Folin, O., and Morris, J. L.: On the Determination of Creatinine and Creatine in 

Urine. Jour.. Biol. Chem., 1914, 17, 469. ■ 
Folin, O., and Wu, H. : A System of Blood Analysis. Jour. Biol. Chem., 1919, 38, 81 . 
Folin, O., and Wu, H.: A System of Blood Analysis "New Method for Determination 

of Sugar." Jour. Biol. Chem., 1919, 38, 106. 



458 BIBLIOGRAPHY 

Folin, O.. and Wu, H.: A System of Blood Analysis. Supplement 1. A Simplified 

and Improved Method for Determination of Sugar. Jour. Biol. Chem., 1920, 41, 

367. 
Friedenwald, J., and Kiefer, R. F. : On the Value of the Quantitative Estimation of 

Dissolved Albumin in the Gastric Contents in the Diagnosis of Cancer of the 

Stomach. Am. Jour." Med. Sci., 1916, 152, 321. 
Gait, H. M., and lies, C. C: A Study of the Boas-Oppler Bacillus. Jour. Path, and 

Bact., 1914-15, 19, 239. 
Gibson, C. L.: The Value of the Differential Leukocyte Count in Acute Surgical 

Diseases. Ann Surg., 1906, 43, 485. 
Giffin, H. Z., and Sanford, A. H.: Clinical Observations Concerning the Fragility of 

Erythrocytes. Jour. Lab. and Clin. Med., 1918-19, 4, 465. 
Graham, G. S. : The Oxidizing Ferment of the Myelocyte Series of Cells and its Demon- 
stration by an Alphanaphthol-pyronin Method. Jour. Med. Res., 1916, 35, 231. 
Graham, G. S.: Benzidin as a Peroxidase Reagent for Blood Smears and Tissues. 

Jour. Med. Res.. 1918, 39, 15. 
Gray, Horace: Cell-counting Technic: A Study of Priority. Am. Jour. Med. Sci., 

1921, 162, 526. 
Greenwald, I.: The Estimation of Non-protein Nitrogen in Blood. Jour. Biol. Chem., 

1918, 34, 97. 
Hamraan, L., and Hirschmann, I. I.: Studies on Blood Sugar. Alimentary Hyper- 
glycemia and Glycosuria as a Test of Sugar Tolerance. Arch. Int. Med., 1917, 

20, 761. 
Harrington, T. F. : Industrial Benzol Poisoning in Massachusetts. Boston Med. and 

Surg. Jour., 1917, 177, 203. 
Harvey, S. C: The Quantitative Determination of the Chlorides in the Urine. Arch. 

Int. Med., 1910, 6, 12. 
Hess, A. F.: A Method of Obtaining Cultures from the Duodenum of Infants. Jour. 

Infect. Dis., 1912, 11, 71. 
Hill, L. W.: Report on Leukocytic Inclusion Bodies. Boston Med. and Surg. Jour., 

1914, 170, 792. 
Hopkins, J. G.: A Method for Standardizing Bacterial Vaccines. Jour. Am. Med. 

Assn., 1913, 60, 1615. 
Howell, W. H.: The Life History of the Formed Elements of the Blood, Especially the 

Red Blood Corpuscle. Jour. Morphol., 1890, 4, 57. 
Howell, W. H.: The Condition of the Blood in Hemophilia, Thrombosis and Purpura. 

Arch. Int. Med., 1914, 13, 76. 
Howland, J., and Marriott, W. McK.: Acidosis Occurring with Diarrhea. Am. Jour. 

Dis. Child., 1916, 11, 309. 
Hurwitz, S. H., and Lucas, W. P.: A Study of the Blood in Hemophilia. Arch. Int. 

Med., 1916, 17, 543. 
Hurwitz, S. H., Meyer, K. F., and Ostenberg, Z. : On a Colorimetric Method of Adjust- 
ing Bacteriologic Media to any Optimum Hydrogen-ion Concentration. Proc. 

Soc. Exper. Biol, and Med., 1915, 13, 24. 
Hurwitz, S. H., Meyer, K. F., and Ostenberg, Z. : A Colorimetric Method for the 

Determination of the Hydrogen-ion Concentration of Biological Fluids, with Special 

Reference to the Adjustment of Bacteriological Culture Media. Johns Hopkins 

Hosp. Bull., 1916, 27, 16. 
Jansky: Isohemagglutination: Recommendation that the Jansky Classification be 

Adopted for Universal Use. Sborn. Klin., 1907, 8, 85. Quoted in Jour. Am. 

Med. Assn., 1921, 78, 130. 
Jenner, L.: A New Preparation for Rapidly Fixing and Staining Blood. Lancet, 

1899, 1, 370. 
Johnson, F. B.:« Filarial Infection — An Investigation of its Prevalence in Charleston, 

S. C. South. Med. Jour., 1915, 8, 630. 
Jousset, A.: Sur une nouvelle methode de recherche du bacille tuberculeux. Bull et 

mem. Soc. Med. des Hop. de Paris, 1903, 20, 23. Also Semaine Med., 1903, 22, 22. 
Karsner, H. T.: Transfusion with Tested Bloods. Jour. Am. Med. Assn., 1918, 70, 769. 
Karsner, H. T.: and Koeckert, H. L. : The Influence of Desiccation on Human Normal 

Isohemagglutinins. Jour. Am. Med. Assn., 1919, 73, 1207. 
Kelly, T. H.: Detection of Small Amounts of Blood. Jour. Lab. and Clin. Med., 

1915-16, 1, 897. 
Kober, P. A.: An Improved Nephelometer-colorimeter. Jour. Biol. Chem., 1917, 

29, 155. 



ORIGINAL ARTICLES 459 

Kolmer, J. A.: Leukocytic "Inclusion Bodies" with Special Reference to Scarlet 

Fever. Am. Jour. Dis. Child., 1912, 4, 1. 
Lambert, S. W., and Patterson, H. S.: Poisoning by Mercuric Chloride and its Treat- 
ment. Arch. Int. Med., 1915, 16, 805. 
Larrabee, R' C: The Volume Index of the Red Corpuscles. Jour. Med. Res., 1911, 

24, 15. 
Lawson, M. R.: Free Malarial Parasites and the Effect of the Migration of the Para- 
sites of Tertian Malarial Infections. Jour. Exp. Med., 1914, 19, 523. 
Lee, R. I.: A Simple and Rapid Method for the Selection of Suitable Donors for 
Transfusion by the Determination of Blood Groups. British Med. Jour., 1917, 2, 
684. 

Lee, R. I., Minot, G. R., and Vincent, B.: Splenectomy in Pernicious Anemia. Jour. 
Am. Med. Assn., 1916, 67, 719. 

Lee, R. I., and Vincent, B.: The Coagulation of Normal Human Blood. Arch. Int. 
Med., 1914, 13, 398. 

Lee, R. I., and White, P. D.: A Clinical Study of the Coagulation Time of Blood. 

Am. Jour. Med. Sc, 1913, 145, 495. 
Levine, Max: Notes on Experiences with the Presumptive and Confirmatory Tests 
for B. coli in Water Analysis for the American Expeditionary Forces. Jour. Am. 
Water Works Assn., 1920, 7, 188. 

Levine, Max: Dysentery and Allied Bacilli. Jour. Infect. Dis., 1920, 27, 31. 

Levy, R. L., Rowntree, L. G., and Marriott, W. McK.: A Simple Method for Deter- 
mining Variations in the Hydrogen-ion Concentration of the Blood. Arch. Int. 
Med., 1915, 16, 389. 

Lewis, R. C, and Benedict, S. R.: A Method for the Estimation of Sugar in Small 
Quantities of Blood. Jour. Biol. Chem., 1915, 10, 61. 

Libman, E.: Clinical Significance of Blood Examinations: The Bacteriology. Inter- 
nal Clin., 1919, 29 S., 3, 125. 

Lord, F. T., Scott, A. C, and Nye, R. N.: Relation of Influenza Bacillus to the Recent 
Epidemic of Influenza. Jour. Am. Med. Assn., 1919, 72, 188. 

Lucas, W. P.: The Value of the Blood Picture in the Early Diagnosis of Measles, 
Especially in Relation to the Question of Isolation. Am. Jour. Dis. Child., 1914, 
7, 149. 

Lundsgaard, C: I. Studies of Oxygen in the Venous Blood. II. Studies of the Oxygen 
Unsaturation in the Venous Blood of a Group of Patients with Circulatory Disturb- 
ances. Jour. Exp. Med., 1918, 27, 179. III. Determinations on Five Patients 
with Compensated Circulatory Disturbances. Jour. Exp. Med., 1918, 27, 199. 
IV. Determinations on Five Patients with Incompensated Circulatory Disturb- 
ances. Jour. Exp. Med., 1918, 27, 219. 

Lyon, B. B. V.: Diagnosis and Treatment of Diseases of the Gall-bladder and Bile 
Ducts. Jour. Am, Med Assn., 1919, 73, 980. 

McLean, F. C: Clinical Determination of the Renal Function by an Index of Urea 
Excretion. Jour. Am. Med. Assn., 1915, 66, 415. 

McLean, F. C: The Numerical Laws Governing the Rate of Excretion of Urea and 
Chlorides in Man. I. An Index of Urea Excretion and the Normal Excretion of 
Urea and Chlorides. Jour. Exp. Med., 1915, 22, 212 and 366. 

McNeil, H. L.: The Use of the Duodenal Catheter in Diagnosis. Am. Jour. Med. 
Sci., 1916, 151, 106. 

MacNeal, W. J., and Chace, A. F.: A Contribution to the Bacteriology of the Duo- 
denum. Arch. Int. Med., 1913, 12, 178. 

MacRobert, R. G.: The Cause of Lumbar Puncture Headache. Jour. Am. Med. 
Assn., 1918, 70, 1350. 

Marriott, W. McK.: The Determination of Alveolar Carbon Dioxid Tension by a 
Simple Method. Jour. Am. Med. Assn., 1916, 66, 1594. 

Marriott, W. McK.: A Method for the Determination of the Alkali Reserve of the 
Blood Plasma. Arch. Int. Med., 1916, 17, 840. 

Marshall, E. K. : A Rapid Clinical Method for the Estimation of Urea in Urine. Jour. 
Biol. Chem., 1913, 14, 283. 

Marshall, E. K.: A New Method for the Determination of Urea in Blood. Jour. 
Biol. Chem., 1913, 15, 487. 

Marshall, E. K. : The Determination of Urea in Urine (Second Communication) . Jour. 
Biol. Chem., 1913, 15, 495. 

Meader, C. N. : Five Cases of Relapsing Fever Originating in Colorado, with Positive 
Blood Findings in Two. Colorado Med., 1915, 12, 365. 



460 BIBLIOGRAPHY 

Meltzer, S. J.: The Disturbance of the Law of Contrary Innervation as a Patho- 
genetic Factor in the Diseases of the Bile Ducts and Gall-bladder. Am. Jour. 
M.d. Sci., lit 17, 153, 469. 

Miller, H. R.: A Review of the Complement-fixation Tcsl in Tuberculosis. Jour. 
Lab. and Clin. Med., 1915-16, 1, 816. 

Miller, H. R., and Zinsser, H.: Complement-fixation Test in Tuberculosis. Proc. 
Soc. Exp. Biol, and Med., 1916, 13, 134. 

Miller, J. A., and Reed, M. A.: Studies of the Leukocytes in Pulmonary Tuberculosis 
and Pneumonia. Arch. Int. Med., 1912, 9, 609. 

Miller, S. R.: The Normal Differential Leukocyte Count. Johns Hopkins Hosp. 
Bull., 1914,25, 317. 

Miller, S. R., and Baetjer, W. A.: Bence-Jones- Proteinuria. Some Observations on 
its Occurrence, with Particular Reference to Nephritis and Hypertension. Jour. 
Am. Med. Assn., 1918, 70, 137. 

Miller, S. R., Brush, N. D., Hammers, J. S., and Felton, L. D.: A Further Study of 
the Diagnostic Value of the Colloidal-gold Reaction, Together with a Method for 
the Preparation of the Reagent. Johns Hopkins Hosp. Bull., 1915, 26, 391. 

Minor, C. L., and Ringer, P. H.: Arneth's Method of Blood Counting, its Prognostic 
Value in Pulmonary Tuberculosis. Am. Jour. Med. Sci., 1911, 141, 638. 

Minot, G. R.: Diminished Blood Platelets and Marrow Insufficiency. A Classifica- 
tion and Differential Diagnosis of Purpura Hemorrhagica, Aplastic Anemia, and 
Allied Conditions. Arch. Int. Med., 1917, 19, 1062. 

Minot, G. R., and Denny, G. P.: Prothrombin and Antithrombin Factors in the 
Coagulation of Blood. Arch. Int. Med., 1916, 17, 101. 

Minot, G. R., and Lee, R. I.: The Blood Platelets in Hemophilia. Arch. Int. Med., 
1916, 18, 474. 

Montgomery, F. H., and Ormsby, O. S.: Systemic Blastomycosis, its Etiology, Patho- 
logic and Clinical Features; the Relation of Blastomycosis to Coccidioidal Granu- 
loma. Arch. Int. Med., 1908, 2, 1. 

Morris, R. S.: Note on the Occurrence of Howell's Nuclear Particles in Experimental 
Anemia of the Rabbit and in Human Blood. Johns Hopkins Hosp. Bull., 1907, 
18, 198. 

Morris, R. S.: The Value of Ehrlich's Triacid Stain in Blood Work. Jour. Am. Med. 
Assn., 1910, 55, 501. 

Mosenthal, H. O.: Renal Function as Measured by the Elimination of Fluids, Salt 
and Nitrogen, and the Specific Gravity of the Urine. Arch. Int. Med., 1915, 16, 
733. 

Mosenthal, H. O., and Lewis, D. S.: A Comparative Study of Tests for Renal Func- 
tion. Jour. Am. Med. Assn., 1916, 67, 933. 

Moss, W. L.: Studies on Isoagglutinins and Isohemolysins. Johns Hopkins Hosp. 
Bull., 1910, 21, 63. 

Murphy, J. B., and Ellis, A. W. M.: Experiments on the Role of Lymphoid Tissue 
in the Resistance to Experimental Tuberculosis in Mice. Jour. Exp. Med., 1914, 
20, 397. 

Musgrave, P.: Cyto-diagnosis: A Study of the Cellular Elements in Serous Effusions. 
Boston Med. and Surg. Jour., 1903, 148, 256. 

Musgrave, P.: Examination of Pleural Fluids with Reference to Their Etiology and 
Diagnostic Value. Boston Med. and Surg. Jour., 1904, 151, 317. 

Musser, J. H. : Study of a Case of Aplastic Anemia. Arch. Int. Med., 1914, 14, 275. 

Myers, V. C: Chemical Changes in the Blood in Disease. Post-Graduate, 1914, 
29, 505; and Jour. Lab. and Clin. Med., 1919-20, 5, 343 and 418. 

Myers, V. C: Simple Methods for the Determination of Nitrogenous Constituents 
in Urine. Post-Graduate, 1914, 29, 737. 

Myers, V. C: Determination of Blood Urea. Post-Graduate, 1914, 29, 505. 

Myers, V. C: Chemical Constitution of the Blood in Health and Disease. VI. 
Total Solids, Total Nitrogen and Chlorides. Post-Graduate, 1915, 30, 35. 

Myers, V. C: A Simple Colorimeter for Clinical Purposes. Jour. Lab. and Clin. 
Med., 1915-16, 1, 760. 

Myers, V. C, and Bailey, C. V.: The Lewis and Benedict Method for the Estimation 
of Blood Sugar, with Some Observations Obtained in Disease. Jour. Biol. Chem., 
1916, 24, 147. 

Myers, V. C, and Fine, M. C: Essentials of Pathological Chemistry, Including 
Description of the Chemical Methods Employed in Medical Diagnosis. Reprinted 
from Post-Graduate, 1913. 



ORIGINAL ARTICLES 461 

Myers, V. C, and Killian, J. A.: Studies on Animal Diastases. I. The Increased 

Activity of the Blood in Diabetes and Nephritis. Jour. Biol. Chem., 1917, 29, 179. 
Myers, V. C, and Lough, W. G.: The Creatinin of the Blood in Nephritis, its Diag- 
nostic Value. Arch. Int. Med., 1915, 16, 536. 
Myers, V. C, and Short, J. J.: The Estimation of Chlorides in Blood. Jour. Biol. 

Chem., 1920, 44, 47. 
Nicoll, M.: Present-day Opinions of the Value of the So-called Inclusion Bodies in 

Scarlet Fever. Arch. Pediat., 1913, 30, 346. 
Opie, E. L. : The Part of Enzymes in Tuberculous Lesions. Proc. 6th Internat. Cong. 

Tuberc, 1908, Part I, p. 255. 
Palmer, W. W., and Van Slyke, D.D.: Studies of Acidosis. IX. Relationship between 

Alkali Retention and Alkali Reserve in Normal and Pathological Individuals. 

Jour. Biol. Chem., 1917, 32, 499. 
Palmer, W. W. : The Colorimetric Determination of Hemoglobin. Jour. Biol. Chem., 

1918, 33, 119. 
Pepper, O. H. P., and Peet, M. M. The Resistance of Reticulated Erythrocytes 

Arch. Int. Med., 1913, 12, 81. 
Petroff, S. A.: Some Cultural Studies on the Tubercle Bacillus. Johns Hopkins Hosp. 

Bull., 1915, 26, 276. 
Petroff, S., A: Serological Studies of Tuberculosis. Am. Rev. Tuberc, 1917-18, 1, 36. 
Poulton, E. P.: The Significance of Alveolar Carbon Dioxide Determinations in the 

Treatment and Prognosis of Diabetes. British Med. Jour., 1915, 2, 392. 
Pratt, J. H. : History of Blood Platelets, Relation to Disease. Johns Hopkins Hosp. 

Bull., 1905, 16, 201. 
Pritchett, I. W., and Stillman, E. G.: The Occurrence of Bacillus Influenza in Throats 

and Saliva. Jour. Exp. Med., 1919, 29, 259. 
Regan, J. C: Anatomic Points Determining the Direction of the Needle and the 

Proper Route for Lumbar Puncture in Children and Adults. Am. Jour. Med. 

Sc, 1919, 157, 83. 
Rehfuss, M. E.: A Modified Gastroduodenal Tube. New York Med. Jour., 1914, 100, 

374. 
Rehfuss, M. E.: A New Method of Gastric Testing, with a Description of a Method 

for the Fractional Testing of the Gastric Juice. Am. Jour. Med. Sci., 1914, 147, 

848. 
Rehfuss, M. E., Bergeim, O., and Hawk, P. B.: Gastro-intestinal Studies. The Frac- 
tional Study of Gastric Digestion. Jour. Am. Med. Assn., 1914, 63, 909. 
Riesman, D.: Pleural Effusion with Inversion of the Diaphragm Producing an Abdom- 
inal Tumor; Together with Remarks on Acute Pulmonary Edema Following 

Tapping. Am. Jour. Med. Sci., 1920, 159, 353. 
Rivas, D., and Smith, A. J.: The Early Diagnosis of Filariasis. South. Med. Jour., 

1912, 5, 631. 
Roberts, D.: A Benzidin Tablet Test for Occult Blood. Jour. Am. Med. Assn., 1915. 

65, 244. 
Robertson, M.: Notes upon Certain Anaerobes Isolated from Wounds. Jour. Path. 

and Bact., 1915-16, 20, 327. 
Robinson, A. S. : A Clinical Study of the Secretions on the Proximal and Distal Sides 

of the Pylorus. Arch. Int. Med., 1917, 19, 220. 
Rose, W. C: A Modified Method for the Clinical Estimation of Pepsin. Arch. Int. 

Med., 1910, 5, 459. 
Rosenow, E. C: The Newer Bacteriology of Various Infections as Determined by 

Special Methods. Jour. Am. Med. Assn., 1914, 63, 903. 
Rothera, A. C. H. : Note on the Sodium Nitro-prusside Reaction for Acetone. Jour. 

Physiol., 1908, 37, 491. 
Rous, Peyton, and Turner, J. R. : A Rapid and Simple Method of Testing Donors for 

Transfusion. Jour. Am. Med. Assn., 1915, 64, 1980. 
Rowntree, L. G., and Fitz, R.: Studies of Renal Function in Renal, Cardiorenal and 

Cardiac Diseases. Arch. Int. Med., 1913, 11, 258. 
Rowntree, L. G., and Geraghty, J. T.: An Experimental and Clinical Study of the 

Functional Activity of the Kidneys by means of Phenolsulphonephthalein. Jour. 

Pharmae. and Exp. Therap., 1910, 1, 579. 
Rowntree, L. G., and Geraghty, J. T. : The Phthalein Test. An Experimental and 

Clinical Study of Phenolsulphonephthalein in Relation to Renal Function in 

Health and Disease. Arch. Int. Med., 1912, 9, 284. 
Ruediger, E. H. : A Comparison of Six Different Antigens in the Wassermann Reac- 
tion- Jour. Infect. Dis., 1919, 24, 31, 



462 BIBLIOGRAPHY 

Ruediger, E. H.: The Influence of Antigen Dilution on the Wassermann Reaction. 
Jour. Infect. Dis., 1919, 25, 224. 

Russell, F. F.: The Isolation of Typhoid Bacilli from Urine and Feces with the Descrip- 
tion of a New Double Sugar Tube Medium. Jour. Med. Res., 1911-12, 25, 217. 

Scott, R. W.: The Effect of the Accumulation of Carbon Dioxide on the Tidal Air, 
and on the H-ion Concentration of the Arterial Blood in the Decerebrate Cat. 
Am. Jour. Physiol., 1917, 44, 196. 

Selling, L. : Benzol as a Leukotoxin. Studies on the Degeneration and Regeneration 
of the Blood and Hematopoietic Organs. Johns Hopkins Hosp. Rep., 1916, 17, 83. 

Selling, L.: A Preliminary Report of Some Cases of Purpura Hemorrhagica Due to 
Benzol Poisoning. Johns Hopkins Hosp. Bull., 1910, 21, 33. 

Shawan, H. K.: The Principle of Blood Grouping Applied to Skin Grafting. Am. 
Jour. Med. Sc, 1919, 157, 503. 

Smith, J. W., and MacNeal, W. J.: Comparative Study of Different Antigens and of 
Different Temperatures of Incubation in the Wassermann Test. Jour. Infect. 
Dis., 1917, 21, 233. 

Smithies, F. : The Significance of Gastric Ulcer with Respect to Gastric Cancer. Jour. 
Am. Med. Assn., 1913, 61, 1793. 

Solis-Cohen, M., and Strickler, A.: The Leukocytic Picture in Pulmonary Tubercu- 
losis. Am. Jour. Med. Sc, 1911, 142, 691. 

Sondern, F. E.: The Present Attitude of Blood Examination for Diagnostic Pur- 
poses. Boston Med. and Surg. Jour., 1903, 153, 690. 

Sondern, F. E.: The Present Attitude of Blood Examination in Surgical Diagnosis. 
Med. Rec, 190.5, 67, 452. 

Stober, A. M.: Systemic Blastomycosis. A Report of Its Pathological, Bacterio- 
logical and Clinical Features. Arch. Int. Med., 1914, 13, 509. 

Suggestions to Medical Examiners by the Insurance Editor, "The Urine." Med. Rec, 
1912, 82, 449, 634, 680, and 1914, 85, 1179. 

Swift, H. F.: A Comparative Studv of Serum Diagnosis in Syphilis. Arch. Int. 
Med., 1909, 4, 376. 

Swift, H. F., and Ellis, A. W. M.: The Cerebrospinal Fluid in Syphilis. Jour. Exp. 
Med., 1913, 18, 162. 

Thayer, W. S. : On Hemolytic Jaundice. Illinois Med. Jour., 1911, 19, 174. 

Thomas, B. A.: Chromocystoscopy in Functional Renal Diagnosis Based upon the 
Employment of Indigocarmin. Surg., Gyn. and Obst., 1909, 8, 368. 

Thomas, B. A.: The Quantitative Determination of Functional Renal Insuffi- 
ciency by the Duboscq Colorimeter; Indigocarmin Versus Phenolsulphonephtha- 
lein. Am. Jour. Med. Sc, 1911, 142, 376. 

Thomas, B. A.: The Value of Chromo-ureteroscopy in Functional Kidney Diagnosis. 
Surg., Gyn. and Obstet., 1911, 12, 345. 

Thomas, B. A.: The Results of Two Hundred Chromo-ureteroscopies Employing 
Indigocarmin as a Functional Kidney Test. Jour. Am. Med. Assn., 1913, 60, 185. 

Thomas, B. A.: The Role of Functional Kidney Tests and Preoperative and Post- 
operative Treatment in the Reduction of Prostatectomy Mortality. Jour. Am. 
Med. Assn., 1914, 63, 1909. 

Thomas, B. A., and Birdsall, J. C. Comparative Results of Various Functional 
Kidney Tests. Jour. Am. Med. Assn., 1917, 69, 1747. 

Thomson, D.: A New Culture Medium for the Gonococcus. British Med. Jour., 
1917, 1, 869. 

Tileston, W., and Griffin, W. A.: Chronic Family Jaundice. Am. Jour. Med. Sci., 

1910, 139, 847. 

Van Giesen, I.: The So-called "Much Granules" in Tuberculosis. Collected Studies 
from the Research Laboratory of the Department of Health, New York City, 

1911, 6, 265. 

Van Slyke, D. D.: Gasometric Determination of the Oxygen and Hemoglobin of 

Blood. Jour. Biol. Chem., 1918, 33, 127. 
Van Slyke, D. D., and Cullen, G. E.: A Permanent Preparation of Urease and its Use 

for Rapid and Accurate Determination of Urea. Jour. Am. Med. Assn., 1914, 

62, 1558. 
Van Slyke, 1). 1)., and Cullen G. E. : The Bicarbonate Concentration of the Blood 

Plasma: its Significance and it- Determination as a measure of Acidosis. 

Jour. Biol. Chem., 1917, 30, 289. 
Van Slyke, I). ]>., and Donleavy, .1. J.: A Simplification of the McLean-Van Slyke 

Method for Determination of Plasma Chlorides. Jour. Biol. Chem., 1919, 37, 55. 



ORIGIN At ARTICLES 463 

Vogel, K. M.: Detection of Mercury in the Excretions. Jour. Am. Med. Assn., 

1914, 62, 532. 
von Wedel, H.: A Contribution to the Study of the Complement-fixation Reaction 

for Tuberculosis. Jour. Immunol., 1918, 3, 351. 
Walker, O. J.: An Index of Body Resistance in Acute Inflammatory Diseases as 

Indicated by Examination of the Blood. Jour. Am. Med. Assn., 1919, 72, 1453, 
Walpole, G. S.: A Method of Titrating Physiological Fluids. Proc. Physiol, Soc 

London, 1910, xxvii. 
Warfield, L. W. : The Differential Leukocyte Count in the Newborn. Am. Med., 

1902, 4, 457. 
Waring, J. J.: Relapsing Fever Endemic in Colorado. Am. Jour. Med. Sci., 1918, 

155, 819. 
Webb, G. B., and Williams, W. W. : Some Hematological Studies in Tuberculosis. 

Trans. Natl. Assn. for Study and Prevention of Tuberculosis, 1909, p. 231. 
West, C. E.: The Bacteriology of Chronic Postnasal Catarrh. Proc. Roy. Soc. Med., 

1910-11, 4, Otol. Sec. 43. 
White, C. Y., and Pepper, W.: Granular Degeneration of the . Erythrocyte. Tr. 

Assn. Am. Phys., 1901, 16, 410. 
Williamson, C. S. : Influence of Age and Sex on Hemoglobin. A Spectrophotometric 

Analysis of Nine Hundred and Nineteen Cases. Arch. Int. Med., 1916, 18, 505. 
Wilson, T. M.: On the Chemistry and Staining Properties of Certain Derivatives of 

the Methylene Blue Group when Combined with Eosin. Jour. Exp. Med., 1907, 

9, 645. 
Wilson, M. A.: A Contribution to the Study of the Complement-fixation Reaction in 

Tuberculosis. Jour. Immunol., 1918, 3, 345. 
Wolff, W., and Junghans, P.: Ueber die quantitative Bestimmung geloster Eiweis- 

stoffe im Mageninhalt. Berl. klin. Wchnschr., 1911, 48, 978. 
Wright, J. H. : The Histogenesis of the Blood Platelets. Jour. Morphol., 1910, 21, 263. 
Yaoita, S.: Ein neues Verfahren zur Auffindung sparlicher Parasiteneier in Feces. 

Deutsche med. Wchnschr., 1912, 38, 1540. 



INDEX. 



Abdomen;, fluid of, examination of, 383 
of breast, blood cultures in, 106 
of liver, blood cultures in, 106 
3, leukocytosis in, 65 
Absorptive power of stomach, 304 
Accidental albuminuria, 222 
Acetic acid in gastric contents, 303 
Acetone in urine, significance of, 215 

tests for, 234 
Achlorhydria, 298 ' 
Achorion schoenleinii, 394 
Achylia, 298 

gastrica, gastric findings in, 305 
pancreatica, duodenal contents in, 
308 
Acid, boric, tests for, 405 

intoxication, 198 
Acidosis, 198 

in diabetes mellitus, 236 ' 
significance of blood tests for, 211 
in starvation, 236 

tests for, tolerance to sodium bicar- 
bonate as, 276 
Actinomycosis in sputum, 355 
Addison-Biermer type of anemia, 76. 

See Pernicious anemia. 
Addisonian anemia, 76. See Pernicious 

anemia. 
'Agar, ascitic-fluid, 426 

Avery's blood-oleate, 368 
bile-salt, MacConkey's, 428 
brilliant-green, Krumwiede's, 429 
double-sugar, Russell's 428 
Endo's fuchsia, 427 
eosine-methylene-blue, Levine's, 428 
hydrocele-ifluid, 426 
Russell's double-sugar, 428 
Agar-agar, blood, 359, 362 
glucose, 426 
meat-extract, 425 
meat-infusion, 425 
Air, alveolar, CO2 tension of, 405 

determination of, Frider- 
icia method, 405 
Marriott's method,407 
significance of findings, 410 
Albumin, dissolved, tests for, in gastric 
contents, 302 
in exudates, determination of, 384 
in urine, method of recording, 222 
qualitative tests for, 220, 242 
30 



Albumin in urine, significance of, 222 
Albuminuria, 222 
accidental, 222 
orthostatic, 222 
renal, 222 
significance of, 222 
types of, 222 
"Alkali reserve," 198, 208 
Altitude, effect of, on erythrocyte count, 

38 
Ambard's cofficients, 273 
Ammonia in urine, 259, 261, 265 
Amoeba coli, 327. See Entamoeba coli. 
dysenterise, 326. See Entamoeba 

histolytica. 
in sputum, 355 
Amylase, tests for, 307 
Anacidity, 298 

Anaerobes, Robertson's medium for, 429 
Anchylostoma duodenale, 341 

infestation with, eosinophilia in, 
65 
Anemia, Addison-Biermer type, 76 
Addisonian, 76 
aplastic, 78 
chlorosis, 75 
classification of, 74 
general consideration of, 74 
hemolytic, chronic, 76. See Perni- 

nicious anemia, 
megaloblastic, 76. See Pernicious 

anemia, 
pernicious, 76 

bile pigments in plasma, 73 
lymphocytosis in, 66 
platelet count in, 41 
resistance of erythrocytes in/ 73 
reticulated erythrocytes in, 69 
volume index in, 43 
primary, 75 
progressive, 76. See Pernicious 

anemia, 
pseudoleukemica infantum, 81 
reticulated erythrocytes in, 69 

resistance of erythrocytes in, 73 
secondary, 79 

due to hemorrhage, 79 
to infections, 79 
to intoxications, 80 
to malignancy, 79 
to poor hygienic surround- 
ings, 81 
occurring in puerperium, 79 



466 



INDEX 



Anemia, secondary, platelet count in, 41 
volume index in, 43 
splenic, lymphocytosis in, 66 

with splenomegaly, 81 
Animal, inoculation of, 401 
Aniscytosis, 52 
Antiformin. preparation of, 441 

use of, in examining sputum, 357 
Anuresis, 21S 
Anuria, 218 _ 

Anus, oxyuric vermicularis about, 324 
Aplastic anemia, 78 
Apparatus, blood chemistry, 445 

for quantitative determination of 

urine, 445, 446 
serology, 447 

for small clinical laboratory, 444 
Appendicitis, blood culture in, 106 
Appleton, reference to, 27 
Arneth formula, S9 
Arsenic poisoning, anemia in, SO 
Artery, superior mesenteric, thrombosis 

of. 324 
Ascaris lumbrocoides, 338 

mystax, 339. See Belascaris cati. 
Ascitic-fluid agar, 426 
Ashford, reference to, 353 
Asthma, bronchial, eosinophilia in, 65 

sputum in, 370 
Austin and Van Slyke's method for deter- 
mination of blood chlorides, 213 
Autogenous vaccines, preparation of, 449 
preservation of, 451 
standardization of, 449 
sterilization of, 450 
Avery, Chickering, Cole and Dochez, 
method for typing 
pneumococci, 362 
reference to, 106 
Avery's blood-oleate agar, 368 

method of cultivating B. influenza?, 
360 
Avoirdupois weight, tables of, 451 



B 



Babcock method for determination of 

fat in milk, 402, 403 , 
Bacilli, Boas-Oppler, in gastric contents, 

298 
Bacillus bulgaricus, identical with Boas- 
I >ppler bacillus, 300 
coli communis, infections with, leu- 
kocytosis in, 65 
in spinal fluid, 387 
diphtheria?, description of, 391 
in ear, 3S9 
in eye, 388 

in mouth and throat, 391 
staining of, 391, 436 
influenzae in spinal fluid, 380 

in sputum, cultivation of, 359 

staining of, 359 
in transudates and exudates, 386 



Bacillus, Koch-Weeks, 388 

maximus buccalis, differentiation of, 

from Boas-Oppler bacillus, 299 
Morax-Axenfeld, 388 
mucosus capsulatus in ear, 389 

in sputum, 369 
pyocyaneus in ear, 389 
in spinal fluid, 381 
smegma? in sputum, 369 
tuberculosis in feces, 320 
inoculation of, 401 
in spinal fluid, 381 
in sputum, 356 

cultivation of, 358 
staining of, 356 
staining of, 436 

in transudates and exudates,386 
in urine, 276 
typhosus in feces, 321 

in spinal fluid, 381 
xerosis, 388 
Bacteria in urine, 218, 230 
Bacteriological examination of dairy 
milk, 405 
of duodenal contents, 309 
of feces, 319 
of spinal fluid, 379 
of transudates and exudates, 384 
of urine, 276 
methods, 411 

apparatus for, 415 

cultural, 411 

direct microscopic examination, 

411 
glassware for, 415 
preparation of media, 414 
staining, 414 
Balantidium coli, 329 

minutum, 329 
Banti's disease, 81 
Barker, reference to, 61, 75, 240 
Bass and Johns method for concentra- 
ting blood for detecting 
malaria organisms, 92 
for cultivating malaria par- 
asites, 98 
ruling of hemocytometers, 33 
Baths, cold, effect of, on leukocvte count, 

39 
Bauer, reference to, 122 
Beef-tapeworm, 332. .Sec Ta-nia sagi- 

nata. 
Belascaris cati, 339 

mystax, 339. 
Bence- Jones protein in urine, 240 
Benedict, A. L., reference to, 296, 297 
Benedict and Lewis, reference to, 169, 

175 
Benedict-Bock formula for Xessler's 

solution, 177 
Benedict, reference to, 163, 225 
Benedict's method for preparing creati- 
nine zinc chloride, 443 
for quantitative determination 
of glucose in urine, 244 



INDEX 



467 



Benedict's qualitative test for glucose in 

urine, 223 
Benzidine reaction for blood in urine, 237 
Benzol poisoning, anemia in, 80 
Beta-oxybutyric acid in urine, tests for, 

235 
Bile acids in urine, 233 

appearance of, in pathological con- 
ditions, 310 
cholecystitis, 310 
choledochitis, 310 
cholelithiasis, 310 
duct, common, obstruction of, feces 

in, 323 
in gastric contents, 292, 295 
medium, Conradi's, 429 

lactose, Jackson's, 429 
obtaining by duodenal tube, 309 
pigments, in blood plasma, 73 
in feces, 315 

in stomach contents, 292, 295 
in urine, significance of, 233 
test for, 233 
Bile-salt sugar, MacConkey's, 428 
Bilharz, reference to, 331 
Bilharziasis, 331 

Bilirubin, crystals of, in urine, 231 
Bismarck brown, 437 
Blackfan's cupping device, 127 
Blake bottles, 415 
Blankenhorn, reference to, 73 
Blastomycetes in sputum, 353 
Bleeding time, determination of, 71 
Blood agar, 359, 362 
bacteriology of, 101 
bile pigments in, 73 
bleeding time of, 71 
carbon-dioxide combining power of, 

200 
casts in urine, 227 
cells, history of, 62 

origin of, 60 
chemical changes in, 168, 196 
examination of, 162 

choice of methods of, 167 
determination of alkali re- 
serve of plasma, 208 
of carbon-dioxide com- 
bining power, 199 
of chlorides, 211 
of creatinine, 187, 188 
plus creatine, 190 
of diastatic activity, 

175 
of non-protein nitro- 
gen, 183, 185 
of oxygen, 215 
of reaction of serum 
to phenolphthalein, 
207 
of sugar, 169, 171 

tolerance, 176 
of urea, 177, 180 
of uric acid, 191, 194 
line of procedure in, 168 



Blood, chemical examination of, ob- 
taining blood for, 167 
routine procedure in, 166 
chemistry, apparatus for, 445 
chlorides of, determination of, 211, 
213 
in malignancy, 215 
in parenchymatous nephritis, 

215 
in pneumonia, 215 
coagulation time of, 70 

determination of, Howell's 
method, 71 
Lee and White method, 70 
simple slide method, 71 
corpuscles, enumeration of, 17, 27 
counting, 27 

apparatus for, 27 
creatinine in nephritis, 190 
diastatic activity of, 175 
erythrocytes, resistance of, to salt 
solution, 72 
reticulated, 69 
vital staining of, 69 
examination of, 17 

clinical significance of, 85 
equipment for, 17 
indications for, 17 
obtaining of, 18 
in feces, 313, 314, 318 
films of, 43 

preparations of, on cover- 
glasses, 43 
on slides, 45 
staining of, 47 
formation of, 59 

theories regarding, 63 
in gastric contents, 295 

significance of, 295 
tests for, 295 
grouping of, 112, 114 
hemoglobin content of, 17 
in infancy and childhood, 67 
matching of, 114 
morphology of, 43 
parasitic diseases of, 91 
pathology of, 74 
plaques. See Platelets, 
platelets of, 39, 55 
prothrombin content of, 71 

determination of, 71 
reactions, microchemical, 68 
serum reactions, 107 

agglutination tests, 107 
for paratyphoid infec- 
tion, 107 
for typhoid infection, 
107 
complement-fixation tests, 
118 
for gonorrhea, 156 
for syphilis, 118 
for tuberculosis, 159 
iso-agglutination tests, 112 
precipitin reaction, 115 



468 



IXDEX 



Blood, specific gravity of, 26 

determination of, 26 
relation to hemoglobin con- 
tent, 26 
staining of, 47 

Ehrlich's method, 50 
Hasting's method, 47 
Leishman's method, 47 
Romanowsky's method, 47 
Wright's method, 47 
stains, study of, 50 
sugar in diabetes mellitus, 175 
tests for acidosis, significance of, 211 
in diabetes, 166 
in gout, 167 
in nephritis, 167 
transfusion of, blood grouping in,l 12 
urea in nephritis, 183 
in urine, 229, 237 

significance of, 238 
tests for, 237 
Blood-flukes, 331 
Blood-oleate agar, Avery's, 368 
Blood-serum, Loeffler's, 426 
Boas-Oppler bacilli in gastric contents, 

299 
Boas's reagent, 293 
test meal, 287 
Bock and Benedict's colorimeter, 164 
Body fluids, examination of, 371 
Boggs and Pincoffs, reference to, 353 
Bone marrow, giant cells of, 58 
tumors, eosinophilia in, 65 
Boric acid, tests for, 405 
Bothriocephalus balticus, 338. See Di- 
bothriocephalus latus. 
latissimus, 338. See Dibothrioce- 

phalus latus. 
latus, 338. See Dibothriocephalus 
latus. 
Bouillon, nutrient. See Broth, nutrient. 
Brain tissue, examination of, for Negri 

bodies, 400 
Breast, abscess of, blood cultures in, 106 
Briggs, reference to, 90 
Brilliant-green agar, Krumwiede's, 429 
Bronchial asthma, sputum in, 370 
Bronchitis, acute, sputum in, 369 

fibrinous, sputum in, 370 
Broth, nutrient, calcium carbonate, 424 
carbohydrate, 424 
meat-extract, 423 
meat-infusion, 423 
sugar-free, 424 
Brown and Petroff, 162 
Brown, James Howard, reference to, 

106 
Buckman and Hallisey, method for 
counting platelets, 39 
reference to, 69 
Bunge's flagella stain, 139 
Bunting, reference to, til 
Burn's method for treponemata, 399 
Bushnell and Treuholtz, reference to, 90 
Butyric acid in gastric contents, 303 



Cabot, reference to, 64, 78 
Cabot's ring-bodies, 53 
Calcium carbonate medium, 424 
in urine, 232 
oxalate, crystals of, in urine, 231 
Capsules, stains for, 438 
Carbohydrate medium, 424 
Carbol-fuchsin for counter-staining, 437 

Ziehl-Neelsen's, 435 
Carbon-dioxide combining power of 

blood, 199 
Carcinoma of intestines, feces in, 324 

of stomach, gastric findings in, 305 
Carlson, reference to, 286 
Castellani, reference to, 111. 
Casts, fibrinous, in sputum, 350 

in urine, 227 
Catarrh of stomach, gastric findings in, 

306 
Catarrhal enteritis, acute, feces in, 323 
Cat-fluke. See Opistorchis fehneus. 
Cercomonas intestinalis. See Lamblia 

intestinalis. 
Cerebrospinous syphilis, Wassermann 

reaction in, 155 
Cestoda, 331 

life history of, 332 
Cestodes, preservation and staining of, 

347 
Chace, reference to, 309 
Chancre, examination of, 396 

soft, organisms in, 399 
Charcot-Leyden crystals in sputum, 352 
Charleston, S. C, an endemic focus for 

filaria infection, 100 
Chatard, Avery, Chickering, Cole and 

Dochez, reference to, 87 
Chauffard-Minkowski type of hemolytic 

jaundice, 84 
Childhood, blood in ,67 
Chlorides of blood, determination of, 21 1, 
213 
in urine, 249, 251, 265 
Chlorosis, 75 

platelet count in, 41 
volume index in, 43 
Cholecystitis, appearance of bile in, 310 

duodenal contents in, 308 
Choledochitis, appearance of bile in, 310 
Cholelithiasis, appearance of bile in, 310 
Cholera, feces in, 322 
leukocytosis in, 65 
Cholesterol crystals in sputum, 353 
Cirrhosis of fiver, lymphocytosis in. (>•> 
( it ion's scale of readings, 150 
"Clap shreds," 218, 393 
Clark, Sir Andrew, method of examining 

sputum, 352, 356 
Clonorchis endemicus, 330 

sinensis, 330 
Coagulation time of blood, 70 
Coefficients, Ambani's, 273 
McLean's. 273 



INDEX 



460 



Cole and Onslow's color-comparator, 418 
Colitis, mucous, feces in, 323 
Colloidal-gold reaction in spinal fluid,375 
Color index, definition of, 41 

method of determining, 41 
Colorimeter, Autenrieth-Koenigsberger, 
268, 269 
Hellige, 268, 269 
Colorimeters, 164, 288, 289 
Colorimetry, 163 

Comparator for colors, Cole and Ons- 
low's, 418 
Complement-fixation reactions, 118, 156, 

159 
Concentration methods for detecting 

malaria parasites in blood, 92 
Concretions in feces, 313 

in sputum, 351 
Congestion of lung, chronic passive, spu- 
tum in, 370 
Conradi's bile-medium, 429 
Craig, reference to, 86, 122, 128 
Craig and Nichols, reference to, 128 
Craig's antigen, 159 
Creatine in urine, 263, 264, 265 
Creatinine in urine, 263, 264, 265 

zinc chloride, preparation of, 443 
of normoblasts, 78 
Crystals in feces, 318 
in sputum, 352 

cholesterol, 353 
fatty acid, 353 
hematoidin, 353 
in urine, 230 
Cultures, incubation of, 412 
inoculation of, 411 
pure, isolation of, 413 
study of, 412 
Cummer's apparatus for venipuncture, 

125 
Curschmann's spirals, 351, 352 
Cylindroids in urine, 228 
Cysticercus bovis, 333 

cellulosse, 334 
Cystine, crystals of, in urine, 231 
Cystitis, laboratory findings in, 281, 

282 
Cysts, echinococcus, in sputum, 355 
Cytological examination of transudates 

and exudates, 383 
Cytology of spinal fluid, 374 



Daland's hematocrit, 42 
Dare's hemoglobinometer, 25 
Dark-field illuminator, 397 
Dermatitis herpetiformis, eosinophilia in, 

65 
Desmoid test of Sahli, 304 
Diabetes insipidus, urine in, 281 
mellitus, acidosis in, 236 
blood sugar in, 175 

tests indicated in, 166 



Diabetes mellitus, diastatic activity of 
blood in, 176 
glucose tolerance test in, 176 
glycosuria in, 225 
urine in, 281 
Diacetic acid in urine, significance of, 235 

tests for, 235 
Diarrhea, Cochin, China, Strongyloides 
intestinalis in, 345 
effect of, on erythrocyte count, 38 
Dibothriocephalus latus, 338 
Digestion, effect of, on leukocyte count, 

39 
Dilatation of stomach, gastric findings 

in, 304 
Diphtheria bacillus, 391. See Bacillus 
diphtherise. 
Dohle inclusion bodies in, 90 
leukocytosis in, 65 
Diplococcus gonorrheae, description of, 
393 
in vagina and urethra, 393 
lanceolatus, 388 
pneumoniae, description of, 388 
in ear, 389 

infection with, blood cultures 
in, 104, 108 
leukocytosis in, 65 
in mouth and throat, 389 
in spinal fluid, 380 
in sputum, 360 

cultivation of, 361, 362 
typing of, 362 _ 

agglutination method, 

365 
Avery's rapid method, 

365 
precipitin method, 366 
in transudates and exudates, 
385 
Dipylidium caninum, 337 
"Dissociation of renal function," 273 
Distoma buski, 330. See Fasciolopsis 
buski. 
conus, 330. See Opisthorchis felin- 

eus. 
hematobium, Bilharz, 331. See 

Schistosomum hematobium. 
japonicum, 330. See Clonorchis 

endemicus. 
lanceolatum, 330. See Opisthorchis 

felineus. 
sibricum, 330. See Opisthorchis 
felineus. 
Dittrich's plugs in sputum, 351 
Dobell, Clifford, reference to, 398 
Dock, reference to, 285 
Dock and Bass' method of search for 

parasitic ova, 325 
Dock's test-meal, 288 
Dohle inclusion granules, 90 
Dorset's egg medium, 427 
Dreyer, reference to, 111 
Drinker, reference to, 69 
Drinker and Hurwitz, reference to, 78 



470 



INDEX 



DuBoscq colorimeter, 164 
Ducrey, reference to, 399 
Duke's method of determining bleeding 

time, 71 
Dunham's peptone solution, 424 
Duodenal contents in achvlia panerea- 
tica, 308 
bacteriology of, 309 
in cholecystitis, 308 
in chronic pancreatitis, 308 
examination of, 306 
findings in morbid con dit ions, 

308 
normal appearance of, 307 
obtaining of, 306 
tests for ferments in, 307 
amylase, 307 
lipase, 308 
trypsin, 307 
tube, for removal of gastric con- 
tents, 290 
Durham fermentation tubes, 415 
Dysentery, feces in, 322 



E 



Ear, Bacillus mucosus capsulatus in, 389 
pyocyaneus in, 389 

bleeding from, in rabbit, 133 

Diplococcus pneumoniae in, 389 

exudate from, examination of, 389 

streptococcus in, 389 
Echinococcus cyst, eosinophilia in, 65 

in sputum, 355 
Ecker and Sasano's method of preparing 

antigen, 142 
Ecker's method of cultivating B. influ- 
enzae, 360 
Eclampsia, leukocytosis in, 65 
Egg-medium, Dorset's, 427 
Ehrlich. reference to, 63 
Ehrlich and Lazarus, reference to. 64 
Ehrlich's diazo reaction, 240 

triple stain, 50 
Einhorn, reference to, 307 

tube. 290 
Elastic fillers in sputum, 352 
Elephantiasis as a result of filariasis, 100 
Ellinger, reference to, 237 
Emerson, reference to, 64 
Endamoeba gingivalis in mouth and 

throat, 389 
Kudo's fuchsin agar, 427 
Entamoeba coli, 327 

histolytica, 326 

hominis, 327 
Enteritis, catarrhal, acute, feces in, 323 
Eosine-methylene-blue agar, Levine's, 

428 
Bosmopbiles, 55, 60, 65, 66 
Eosinophilia^ 65, 66 

in trichinosis, 345 
Eosinophilic leukocytes in transudates 
and exudates, 385 



Epididymitis, eosinophilia in, 65 
Epistaxis. anemia in, 79 
Epithelial casts in urine, 227 

cells in urine, 229 
Equivalents of metric and common sys- 
tems, 452 
Erysipelas, blood cultures in, 106 

leukocvtosis in, 65 
Erythemia, 38 

Erythrocytes, count of, physiological 
variations in, 38 
variations in disease, 38 
description of, 52 
enumeration of, 27 
apparatus for, 27 
normal findings, 37 
procedure, 33 
normal number of, 37 
nucleated, 53 

resistance of, to hypotonic salt solu- 
tion, 72 
skeined, 69 
in urine, 229 
volume of, 42 
volume-index of, 43 
Esbach's method for determining albu- 
min in urine. 242 
Euchlorhvdria, 298 
Ewald's t'est meal, 287 
Exanthemata, diagnosis of, 86 
Exercise, effect of, on leukocyte count, 39 
Exophthalmic goiter, lymphocytosis in, 

66 
Exudates, albumin in, determination of, 
384 
examination of, 383 
purulent, examination of, 386 
Eye, exudate from, examination of, 388 



Fasciola hepatica, 329 
Fasriolopsia buski, 330 
Fat, drops of, in feces, 317 

in gastric contents, 298 
Fatty acid, crystals of, in feces, 317 
in gastric contents, 298 
in sputum, 353 
volatile, in gastric contents, 303 
casts in urine, 227, 228 
Favus, 394 

Feces in acute catarrhal exteritis, 323 
bacteriological examination of, 319 
tubercle bacilli, 320 
typhoid bacilli, 321 
in carcinoma of intestines, 324 
in cholera, 322 
composition of, 311 
chemical examination of, 314 
bile pigments, 315 
blood, 314 
reaction, 314 
in diseases of pancreas, 323 
in dysentery, 322 



INDEX 



471 



Feces, examination of, -311 

apparatus required, 311 
routine procedure for, 311 
in gastro-enteritis, 322 
in intussusception, 324 
. method of transporting, 3 12 
macroscopic examination of, 312 
blood, 313 
color, 312 
concretions, 313 
consistency, 312 
form, 312 
mucus, 313 
odor, 313 
parasites, 314, 324 
pus, 312 
microscopic examination of, 316 
blood, 318 
crystals, 318 
food remnants, 316 
mucus, 318 
parasitic ova, 319 
pus, 318 
in mucous colitis, 323 
in obstruction of common bile duct, 

323 
test diet for examination of, 321 
in typhoid fever, 321, 322 
in ulcers of intestines, 324 
Fehling's method for quantitative deter- 
mination of glucose in urine, 243 
reaction with spinal fluid, 374 
test for glucose in urine, 223 
Femoral artery, bleeding from, in.rabbit, 

135 
Fermentation test for sugar in urine, 224 
tubes, 415 

Durham's, 415 
Fever, eosinophilia following, 65 

relapsing, 101 
Fibrin casts in urine, 228 
Fibrinous bronchitis, sputum in, 370 
Filaria, 99 

bancrofti, 99 
diurna. See F. loa. 
embryos of, in sputum, 355 
loa, 100 
medinensis, 99 
perstans, 99 
Filariasis, 99 

eosinophilia in, 65 
Fish-tapeworm, 338. See Dibothrioce- 

phalus latus. 
Flagella, stains for, 439 
Florence reaction, 400 
Fluids, abdominal, examination of, 383 

thoracic, examination of, 383 
Flukes, 329. See Trematodes. 

preservation and staining of, 347 
Folin, reference to, 163, 254, 268, 443 
Folin and Denis, reference to, 166 

uric acid reagent, 191 
Folin and Doisy, reference to, 442 
Folin and Wu's formula for Nessler's 
solution, 180 



Folin aud Wu's methods of determining 
blood creatine 
plus creatinine, 
190 
creatinine, 187 
sugar, 171 
urea, 180 
uric acid, 191 
for preparation of urease, 
177 
Formaldehyde, tests for, 404 
Francis, reference to, 100 
Fridericia method for determining C0 2 

tension of alveolar air, 405 
Friedenwald and Kieffer, reference to, 303 
Friedlander's bacillus, 369. See Bacillus 

mucosus capsulatus. 
Function of kidneys, tests for, 267, 268, 
271, 272, 273 



Gall-stones, chemical examination of, 

324 
Gait and lies, reference to, 300 
Gangrene of lung, sputum in, 370 
Gastric contents, acetic acid in, 303 

acidity of, normal results, 297 

variations in, 298 
albumin (dissolved) in, 302 
Boas-Oppler bacillus in, 299 
butyric acid in, 303 
characteristics of, 287 
crystals of fatty acid in, 298 
examination of, 285 

apparatus required for,285 
physiological considera- 
tions of, 285 
relative simplicity of, 285 
routine procedure for, 291 
test meals for, 287 
Boas's, 287 
Dock's, 288 
Ewald's, 287 
Riegel's, 287 
Salzer's, 288 
fat drops in, 298 
fatty acids, volatile in, 303 
findings in diseased states, 304 
gross appearance of, 291 
amount of, 292 
color of, 292 
layers of, 292 
mucus of, 292 
odor of, 292 
mercury in, 303 
microscopical examination of, 
298 
findings in, 298, 299 
pepsin in, 300 

qualitative test for, 300 
quantitative tests for, 300, 
301 
Mett's, 300 



472 



INDEX 



Gastric contents, pepsin in, quantitative 
tests for, Rose's, 301 
qualitative chemical tests for, 
293 
bile, 292, 295 
blood, 295 
free hvdrochloric acid, 

293 
lactic acid, 293 
quantitative chemical tests for, 
295 
combined acidity, 297 
free hvdrochloric acid, 

295 
hydrochloric acid de- 

ficit, 297 
total acidity, 296 
removal of, contraindications 
to, 289 
for examination, 288 
fractional method for, 290 
with duodenal tube, 290 
sarcinae in, 298 

tumor tissue in, 303 
yeast cells in, 298 
findings in achylia gastrica, 305 
in carcinoma of stomach, 305 
in chronic catarrh of stomach, 

306 
in continuous hypersecretion of 

stomach, 305 
in dilatation of stomach, 304 
in gastrosuccorrhea, 305 
in neuroses of stomach, 308 
in ulcer of stomach, 304 
juice, characteristics of, 287 

physiology of secretion of, 285 
ulcer, anemia in, 79 
Gastro-enteritis, feces in, 322 
Gastrosuccorrhea, gastric findings in, 305 
Gaucher 's disease, 81 
Gay, reference to, 107 
Gelatin, meat-extract, 425 

meat-infusion, 425 
Gentian-violet, anilin-water solution, 436 

Sterling's solution, 436 
Geraghty and Rowntree's method for 

determining renal function, 268 
Gerhardt's test for diacetic acid in urine, 

235 
Giant cells of bone marrow, 58 
Gibson's curve, 87, 88 
Giemsa's stain, preparation of, 441 

for treponemata, 398 
Giffin and Sanford's method of determin- 
ing resistance of erythrocytes to hypo- 
tonic salt solution, 72 
Gilbert, reference to, 73 
Glass-ware, cleaning of, 448 
Glossina morsitans, 99 

palpalis, 99 
Glucose agar, 426 

renal threshhold for, 177 
tolerance test for, 176 

in diabetes mellitus, 176 



Glucose in urine, qualitative tests for, 

223 
Glycosuria, significance of, 225 
Glycuresis, 225 
Gmehn's test, 73 

for bile pigments in urine, 233 
Goiter, exophthalmic, lymphocytosis in, 

66 
Gonococci, Thomson's medium for, 426 
Gonococcus. See also Diplococcus gon- 
orrhea, 
infection with, blood cultures in, 104 
complement-fixation test for, 
156 
strains of, 156 
Gonorrhea, complement-fixation tests 
for, 156 
shreds in urine in, 218, 393 
Gout, blood tests indicated in, 167 
Gradwohl, reference to, 122 
Gram's iodine solution, 437 
stain, 436 

results with, 437 
Granger and Pole's modification of Man- 
son's stain, 90 
Granular casts in urine, 227 
Granules, inclusion, of Dohle, 90 
"Green-sickness," 75 
Grawitz's punctate basophilia, 53 
Greenwald, reference to, 183 
Guinea-pig, bleeding from heart, 139 

from neck, 138 
Gunning's test for acetone in urine, 234 
Giinzberg's reagent, 293 



Halberstadter and Prowazek's tra- 
choma bodies, 389 

Hammann and Hirschman, test for glu- 
cose tolerance, 176 

Hammerschlag's table for ascertaining 
hemoglobin content from specific 
gravity of blood, 26 

"Hanging drop" preparation, 411 

Harrington, reference to, 80 

Hart's test for beta-oxybutyric acid in 
urine, 235 

Hasting's stain, 47 

Hayem, reference to, 41 

Hayem's diluting fluid, 33 

Hayem- Widal type of hemolytic jaun- 
dice, 82 

Hay's test for bile acids in urine, 233 

Heart, bleeding from, in guinea-pig, 139 
decompensation of, oxygen unsatu- 

ration in, 216 
disease of, effect of, on erythrocyte 
count, 38 

"Heart-failure cells," 351 

Hecht -Weinberg, reference to, 122 

Heller's test, for albumin in urine, 220 

Hematocrit, Daland's, 42 

Hematoidin crystals in sputum, 353 



INDEX 



473 



Hematoidin crystals in urine, 231 
Hematoporphyrin, 238 
Hematuria, 229, 237 
Hemocytometers, 27 
chambers of, 29, 31 

rulings of, 32 
pipettes, cleaning of, 35 
Hemoglobin, amount of, at different 
ages, 27 
Appleton's findings, 27 
Williamson's findings, 
27 
determination of, 17, 19 

methods for, comparison of, 19 
Dare's, 25 

Miescher-Fleischl's, 24 
Palmer's colorimetric, 22 
Sahli's, 19 
Tallquist's, 24 
Von Slyke's gasometnic, 20 
in infancy and childhood, 27, 67 
reduced, 238 
Hemoglobinometers, Dare's, 25 
Meischer-Fleischl's, 24 
Sahli's, 19 
Hemoglobinuria, 237 
significance of, 238 
tests for, 237 
Hemolytic pernicious anemia, chronic, 76 
Hemophilia, anemia in, 79 

coagulation time of blood in, 70 
platelet count in, 41 
prothrombin in, 71, 72 
Hemorrhage as a cause of anemia, 79 
leukocytosis in, 65 
platelet count in, 41 
Hemorrhoids, anemia in, 79 
Henderson, reference to, 198 
Hensel, Weil and Jelliffe, reference to, 314 
Herpes zoster, eosinophilia in, 65 
Herzfehlerzellen, 351 
Hess, reference to, 309 
Hippuric acid, crystals of, in urine, 231 
Hiss and Zinsser, reference to, 106 
Hiss's capsule stain, 438 

inulin serum-water, 424 
Hodgkin's disease, large mononuclear 

leukocytosis in, 66 
Holt's apparatus, 402 
Hook-worm, New World species, 343. 
See Necator americanus. 
Old World species, 341 . See Anchy- 
lostoma duodenale. 
Hopkins's method for standardization of 

autogenous vaccines, 450 
Howell's method of determining coagu- 
lation time of blood, 70 
prothrombin content of 
blood, 71 
nuclear particles, 53 
Huntoon's spore stain, 439 
Hurwitz, Meyer and Ostenburg method 

for adjusting reaction of media, 417 
Hyaline casts in urine, 227 
Hydatid disease, 335 



Hydrocele-fluid agar, 426 
Hydrochloric acid deficit in gastric con- 
tents, 297 
free, in gastric contents, 293, 295 
Hydrogen-ion concentration of media, 

417 
Hydrometer for testing urine, 219 
Hymenolepis dimunata, 337 

nana, 335 
Hyperacidity, 298 
Hyperchlorhydria, 298 
Hyperleukocytosis, 39, 64 
Hypersecretion of stomach, continuous, 

gastric findings in, 305 
Hypomycetes in sputum, 353 



Illuminator, dark-field, 397 
"Impression method" of making prepa- 
rations of sputum, 355 
Inclusion granules of Dohle, 90 
"Index of elimination," of Thomas, 272 

of resistance of Walker, 88 
India-ink method for treponemata, 399 
Indican in urine, significance of, 237 

tests for, 236 
Indigo-carmine, excretion of, as test for 

renal function, 271 
Infancy, blood in, 67 
Infections, acute, leukocytosis in, 65 

chronic, lymphocytosis in, 65 
Influenza, lymphocytosis in, 65 
Insocopy, 386 

Insurance, life, urine examination in, 28 1 
Intestines, carcinoma of, feces in, 324 

intussusception of, feces in, 324 

ulcers of, feces in, 324 
Intoxication, acid. See Acidosis. 
Intraperitoneal injections of rabbit, 131 
Intravenous injections of rabbits, 131 
Intussusception of bowels, feces in, 324 
Inulin serum-water, 424 
Irritation forms of Turck, 58 

serum, 396 
Isohemagglutinins, 112 



Jackson's lactose-bile medium, 429 
v. Jaksch's disease, 81 
Jansky, reference to, 112, 113, 114 
Jaundice, chronic, anemia in, 80 

obstructive, resistance of ery- 
throcytes in, 73 
coagulation time of blood in, 70 
hemolytic, 82 

acquired type, 82 
Chauffgard-Minkowski type, 84 
familial type, 84 
Hayem-Widal type, 82 
resistance of erythrocytes in, 73 
reticulated erythrocytes in, 69 



474 



INDEX 



.Tenner's stain, preparation of, 411 
Johnston, reference to, 100 
Jousset, reference to, 386 



Karsxek and Koeehert, reference to, 113 
Keidel's tube, 125 
Kelling's test for lactic acid, 293 
Kelly. T. H., reference to, 315 
Kidney, amyloid, laboratory findings in, 
279 
diseases of, classification of, 278 

findings in, 278 
passive congestion of, 280, 282 
stone of, laboratory findings in, 280 
Kidneys, function of, dissociation of, 273 
methods of determining, 267, 
268, 271, 272, 273 
Killian, reference to, 175 
Kjeldahl's method for determining total 

nitrogen in urine, 252 
Kober's colorimeter, lfi-1 
Koeehert, reference to, 113, 122, 128 
Kolmer, reference to, 91, 118, 159 
Kowarsky's method for phenylhydra- 

zine reaction, 225 
Krumwiede's brilliant-green agar, 429 



Lactic acid in gastric contents, 293 
significance of, 294 
tests for, 293, 294 
Lactose in milk, determination of, 403 
in urine, qualitative tests for, 226 
Lambert and Patterson, reference to, 266 
Lamblia duodenalis, 328 

intestinalis, 328 
Lange colloidal-gold test, 375 

findings, significance of, 

378 
performance of reaction, 

378 
preparation of, 377 

of solutions, 376 
reading reaction, 378 
reagents required, 376 
solutions required 375 
Lateral sinuses, infection of, blood cul- 
tures in, 106 
Lead poisoning, anemia in, 80 
Lee and Vincent, reference to, 40, 70 
Lee and Vogel, reference to, 303 
Lee and White's method of determining 

coagulation time of blood, 70 
Lee, Minot and Vincent, reference to, 70 
Lee's method for blood grouping, 113 
Legal's test for acetone in urine, 234 
Irishman's stain, 47 
Leptothrix buccalis in mouth and throat, 

389 
Leucine, crystals of, in urine, 231 



Leucine in sputum, 352 
Leukanemia, 81 
Leukemia, S4 

lymphatic, acute, 85 
chronic, 85 
lymphocytosis in, 65 
myelogenous, acute, 85 

chronic, 84 
myeloid, neutrophilic leukocytosis 

in, 65 
platelet count in, 41 
urine in, Bence-Jones protein of, 241 
Leukocyte casts in urine, 227 
Leukocytes in chlorosis, 76 

differential count of, 51, 63 

in infancy and childhood, 

67 
normal findings, 64 
enumeration of, 38 
eosinophilic, 55, 60 

in transudates and exudates, 385 
in feces, 312, 318 
immature, 58, 59 
large mononuclear, 57, 60, 65, 66 
of lymphadenoid origin, 59 
lymphocytic, 55 
myeloblastic, 58 
myelocytic, 58 
normal number of, 39 
number of, in infancy and childhood, 
39, 67 
variations in, 39 
polymorphonuclear basophilic, 56, 
60, 65, 66 
eosinophilic, 55, 60, 65, 66 
neutrophilic, 55, 60, 65, 66 
in pus, 386 

in transudates and exu- 
dates, 385 
reaction of, with oxydase stain, 68 
transitional, 57 
in urine, 229 
Leukocytosis, 39 
absolute, 64 
forms of, 64 
Gibson's curve, 87, 88 
interpretation of, 86 
large mononuclear, 66 
lymphocytic, 65, 66 
polymorphonuclear basophilic, 65,66 
eosinophilic, 65, 66 
neutrophilic, 65. 66 
relative, 64 

Sondern's hypotheses regarding, 87 
Walker's index, 88 
Leukopenia, 64 

Le vine's eosine-methylene-blue agar, 428 
Levy chamber for blood counting, 29 
Levy, Magnus, reference to, 235 
Lipase, tests for, 308 
Liquor sodae chlorinatav, 442 
Litmus milk for medium, 424 
Liver, abscess of, blood cultures in, 106 

cirrhosis of, lymphocytosis in, 66, 
Liver-fluke, 329. See Fasciola hepatica. 



INDEX 



475 



Loeffler's blood serum, 426 

methylene-blue, 435 
Lohnstein's saccharometer, 243 
Lord, Scott, and Nye, reference to, 369 
Lundsgaard, reference to, 215 
Lung, chronic passive congestion of, 
sputum in, 370 

disease of, effect of, on erythrocyte 
count, 38 

gangrene of, sputum in, 370 

malignant disease of, 370 

tuberculosis of, aputum in, 370 
Lung-fluke, 355 
Lymphatic leukemia, acute, 85 

chronic, 84 
Lymphoblasts, 59 
Lymphocytes, 56, 62, 65, 66 

mitochondria in, 70 

relative proportions of, in infancy 
and childhood, 67 

in transudates and exudates, 385 
Lymphocytosis, 65 

Lymphosarcoma, large mononuclear leu- 
kocytosis in, 65 
Lyon, reference to, 309, 310 



M 



McLean and Van Slyke, reference to, 213 
McLean's coefficients, 273 
McNeil, reference to, 308 
McPhedran, reference to, 75 
MacConkey's bile-salt agar, 428 
MacLeod, reference to, 236 
MacNeal, reference to, 309 
MacRae's needle, 124 
MacRobert, reference to, 373 
Macrocytes, 52 
Magenche, reference to, 175 
Magnesium sulphate test for albumin in 

urine, 224 
Malaria, 91 

anemia in, 79 

benign tertian, 94 

estivo-autumnal, 95 

large mononuclear leukocytosis in, 66 

parasite of, 92 

development of, 93 

quartan, 95 
Malignancy, anemia in, 79 

blood chlorides in, 215 
Mallory, reference to, 64 
Maltose in urine, qualitative tests for, 

226 
Manson's stain, 90 

Marriott's method for determining al- 
kali reserve of blood plasma, 208 
Marshall, reference to, 177 
Marshall's method for determining urea 

in urine, 256 
Mast cells, 56, 60, 65, 66 
Mastoiditis, blood cultures in, 108 
Meader, reference to, 101 
Measles, Dohle inclusion bodies in, 90 



Measles, lymphocytosis in, 65 
Meat, medium, Robertson's, 429 
Meat-extract agar, 425 

broth, 423 

gelatin, 425 
Meat-infusion agar, 425 

broth, 425 

gelatin, 425 
Media, clearing of, 420 

filtering of, 421 

formulae for, 423, et seq. 

inoculation of, 411 

preparation of, 414 

reaction of, 415 

slanting of, 422 

sterilizing of, 422 

storing of, 423 

tubing of, 421 
Medium, agar, ascitic-fluid, 426 

Avery's blood-oleate, 368 
blood, 359, 362 
Endo's fuchsin, 427 
glucose, 426 
hydrocoele fluid, 426 
meat-extract, 425 
meat-infusion, 425 

blood-serum, Loeffler's, 426 

calcium carbonate, 424 

carbohydrate, 424 

Conradi's bile, 429 

Dorset's egg, 42 

Dunham's peptone, 424 

Endo's fuchsin agar, 427 

gelatin, meat-extract, 425 
meat-infusion, 425 

Hiss's inulin serum-water, 424 

Jackson's lactose-bile, 429 

Krumwiede's brilliant-green agar, 
429 

Levine's eosine-methylene-blue agar, 
428 

Loeffler's, 426 

litmus milk, 424 

MacConkey's bile-salt agar, 428 

meat-extract broth, 423 
gelatin, 425 

meat-infusion broth, 423 
gelatin, 425 

milk, 424 

Petroff's, 426 

potato, 427 

Robertson's cooked-meat, 429 

Russell's double-sugar agar, 428 

serum-water, Hiss's inuhn, 424 

sugar-free broth, 424 

Thomson's, for gonococcus, 426 
Megakaryocytes, 58 
Megaloblastic pernicious anemia, 76 
Megaloblasts, 54 
Megalocytes, 52 
Megastoma entericum. See Lamblia 

intestinalis. 
Meltzer, reference to, 309 
Meninges, tuberculosis of, 381, 382 
Meningitis, blood cultures in, 106 



476 



INDEX 



Meningitis, influenzal. 380 

leukocytosis in, 65 
meningococcus, 380, 382 
pneumococcus 380, 3S2 
tuberculous, 381, 382 
Meningococcus. See Micrococcus intra- 
cellularis meningitidis, 
infections with, leukocytosis in, 65 
Mercury in gastric contents, 303 
poisoning, anemia in, 80 

blood-sugar findings in, 175 
urine in, 266 
Methemoglobin, 238 
Methylene-blue, Loeffler's, 435 
Metric system, tables of, 451 
Mett's method for determining pepsin, 

300 
Microblasts, 54 

Micrococcus catarrhalis in sputum, 367 
in urethra, 393 
intracellulars meningitidis in spinal 

fluid, 380 
tetragenous in spinal fluid, 381 
Microcytic 52 

Miescher-Fleischl's method for determin- 
ing hemoglobin, 24 
Milk, dairy, bacteriological examination 
" of, 405 
examination of, 403 
human, examination of, 402 
lactose in, determination of, 403 
litmus, for medium, 424 
preservatives in, tests for, 404 
protein in, determination of, 403 
Miller, reference to, 64 
Miller and Baetjer, reference to, 241 
Miller and Reed, reference to, 90 
Miller and Zinsser's antigen, 160 
Miller, Brush, Hammers and Felton, 

reference to, 175 
Minor and Ringer, reference to, 90 
Minot, reference to, 78 
Minot and Lee, reference to, 41 
Mitochondria, 70 
Moeller's spore stain, 439 
Molvbdate-phosphate reagent of Folin 

and Wu, 172 
Monilia in sputum, 353 
Montgomery and Ormsbv, reference to, 

353 
Mosenthal test-meal for renal function, 

272 
Mosquito, anopheles, 91, 97 

culex, 98 
Moss, reference to, 112, 113 
Mouse, autopsy of, for typing pneumo- 
cocci, 364 
inoculation of, for typing pneumo- 
cocci, 363 
Mouth and throat, exudates in, exami- 
nation of, 389 
Bacillus diphtheria 1 in, 391 
Diplococcus pneumoniae in, 389 
Endomoeba gingivalis in ,389 
Leptothrix buccalis in, 389 



Mouth and throat, Oidium albicans in, 
389 
Spirocheta buccalis in, 389 

dentium in, 389 
staphylococcus in, 389 
streptococcus in, 389 
syphilis of, 390 
Treponema pallidum in, 390 
Much's granules in sputum, 357 
Mucous colitis, feces in, 323 
shreds in urine, 218 
threads in urine, 229 
Mucus in feces, 318 
Murphy and Ellis, reference to, 57 
Musgrave, P., reference to, 384, 386 
Musser, reference to, 78 
Myeloblasts, 58 
Myelocytes, 58 
Myelogenous leukemia, acute, 85 

chronic, 84 
Myeloma, multiple, Bence-Jones protein 

in urine of, 241 
Myers and Bailey, reference to, 175 
Myers and Bailey's method for determin- 
ing blood sugar, 169 
Myers and Fine, reference to, 307 
Myers and Killian, reference to, 175 
Myers and Lough's method for deter- 
mining blood creatinine, 187 
Myers and Short's method for deter- 
mination of blood chlorides, 212 
Myers' colorimeter, 164 

colorimetric method for determin- 
ing urea in urine, 259 
method for determining blood non- 
protein nitrogen, 183 
urea, 177 
modification of Benedict's method 
for quantitative determination of 
glucose in urine, 245 
reference to, 163, 254, 264 
sugar tube, 170 



N 



Xaegeli, reference to, 63 
Necator americanus, 343 
Xeck, bleeding from, in guinea-pig, 138 

in rabbit, 135 
Necrotic tissue in sputum, 351 
Negri bodies in brain, 400 
Neisser's stain for diphtheria bacilli, 436 
Nematoda, 338 

Nephritis, acute, laboratorv findings in, 
279, 282 
blood creatinine in, 190 
tests indicated in, 167 
urea in, 183 
chemical blood findings in, 196, 197 
chronic, anemia in, SO 

interstitial, albumin and casts 
in, 226 
laboratorv findings in, 279, 
282 



INDEX 



477 



Nephritis, chronic parenchymatous, 
blood-sugar findings in, 
175 
laboratory findings in, 279, 
282 
parenchymatous, blood chlorides in, 
215 
Nephrolithiasis, laboratory findings in, 

280 
Nervous system, central, syphilis of, 

diagnosis of, 155 
Nessler's solution, 177 

Benedict-Bock formula for, 177 
Folin and Wu's formula for, 180 
Neubauer ruling of hemocytometers, 32 
Neuroses, gastric, gastric findings in, 306 
Newton's rings, 30 
Nicoll and Hill, reference to, 91 
Nitric acid ring test for albumin in urine, 

220 
"Nitrogen partition," 265 

total, in urine, 251, 255, 265 
in urine, partition of, 265 
Noguchi, reference to, 122, 127, 142 
Noguchi's test for globulin, in spinal 

fluid, 374 
Nonne's test, 375 
syndrome, 373 
Normoblasts, 54 
crises of, 78 
Nose, exudates of, examination of, 392 
Nuclear particles of Howell, 53 
Nucleated red blood cells, 53, 54 
Nucleoprotein in urine, 240 



Obermayek's test for indican, 236 

Oi'dium albicans in mouth and throat, 389 

Oligocythemia, 38 

Oliguresis, 218 

Oliguria, 218 

Opisthorchis felineous, 330 

Ornithodorous moubata, 101 

Orthostatic albuminuria, 222 

Osier's disease, 38 

Otitis media, blood cultures in, 106 

Ottenburg, reference to, 121 

Oxalic acid solution, preparation of, 433 

"Oxygen unsaturation," 216 

Oxyhemoglobin, 238 

Oxylase granules, 63, 68 

reaction, Graham's, 68 
Oxyuris vermicularis, 340 

about anus, 324 

infestation with, eosinophilia 
in, 65 



Palmer and Van Slyke, reference to, 276 
Palmer's colorimetic method for deter- 
mining hemoglobin, 22 



Pancreas, disease of, feces in, 323 
Pancreatitis, chronic, duodenal contents 

in, 308 
Pandy's test, 375 
Pappenheim, reference to, 63 
Pappenheim's decolorizing fluid, 435 
method of staining for tubercle 
bacillus, 357 
Paracentesis abdominis, 383 

thoracis, 383 
Paragonimus westermannii, description 
of, 355 
ova of, in sputum, 355 
Paramecium coli, 339. See Balantium 

coli. 
Parasites in feces, 314, 324 
ova of, 319 
intestinal, anemia in, 79 
preservation and staining of, 347 
in sputum, 351, 355 
Paratyphoid fever, agglutination tests in, 

107 
Parenchymatous nephritis, chronic, 

blood-sugar findings in, 175 
Pasteur, reference to, 400 
Pea globulin for determining pepsin, 301 
"Pea-soup" stools, 322 
Pearce, Krumbhaar and Frazier, refer- 
ences to, 82, 83 
Pearce, reference to, 69, 82 
Pediculus capitus, 398 
inguinalis, 396 
vestimenti, 396 
Pemphigus, eosinophilia in, 65 
Pepsin in gastric contents, qualitative 
tests for, 300 
quantitative tests for, 300 
Peptone solution, Dunham's, 424 
Peritonitis, blood cultures in, 106 
Pernicious anema, 76 

Addison-Biermer type of, 76 
Addisonia, 76 

bile pigments in plasma, 73 
chronic hemolytic, 76 
erythrocytes in, resistance of,73 
lymphocytosis in, 66 
megaloblastic, 76 
progressive, 76 
volume index in, 43 
Pertussis, lymphocytosis in, 65 
Petri dishes, 415 
Petroff's antigen, 160 
medium, 426 

method of cultivating tubercle bacil- 
lus, 358 
Phenolsulphonephthalein, excretion of, 

as test for renal function, 268 
Phenylhydrazine reaction test for sugar 

in urine, 224 
Phosphate, ammonium-magnesium, crys- 
tals of, in urine, 231 
neutral calcium, plates of, in urine, 

231 
"triple." See Ammonium-magne- 
sium. 



478 



INDEX 



Phosphates in urine, 218, 230. 251 
Picric acid, purification of, 442 

testing of, 442 
"Pin-worms," 340. See Oxyuris vermic- 

ularis. 
Plaques. See Platelets. 
Plasmodium of malaria, 92 
cultivation of, 98 
differentiation of types, 93 
Plasmodium falciparum, 95 
malaria, 95 
vivax, 94 
Platelets in chlorosis, 76 
enumeration of, 39, 55 

Buckman and Hallisey's 

method, 40 
Wright and Kinnicutt's 
method, 39 
number of, in diseased conditions, 41 
in health, 41 
Pleocytosis, 374 

Pneumonia, acute lobar, sputum in, 370 
blood chlorides in, 215 
chlorides in urine in, 251 
Dohle inclusion bodies in, 90 
platelet count in, 41 
Poisoning, arsenic, anemia in, 80 
benzol, anemia in, 80 
lead, anemia in, 80 
mercury, anemia in, 80 

blood-sugar findings in, 175 
urine, in, 266 
Polariscope, use of, for determining 

amount of sugar in urine, 248 
Poliomyelitis, acute anterior, spinal fluid 

in, 382 
Pollitzer bag, 288 
Polychromasia, 52 
Polychromatophila, 52 
Polycythemia, 38 

Polymorphonuclear neutrophilic leuko- 
cytes in transudates and exudates, 385 
"Polys.," 55 
Polyuria, 218 
Potato medium, 427 
Poulton, reference to, 405 
Pratt, reference to, 41 
Precipitin reactions in blood, 115 
Pregnancy, effect of, on leukocyte count, 
3d' 
pyelitis of, blood cultures in, 105 
Preservatives in milk, tests for, 404 
Pritchett and Stillman, reference to, 369 
Prostate, secretion of, examination of,278 

massage of, 278 . 
Protein bodies in urine, qualitative tests 
for, 220 
quantitative tests for, 242 
in milk, determination of, 403 
Proteins, precipitin reactions for, 117 
Prothrombin content of blood, 71 

in purpura hemorrhagica, 
72 
Provocative test in syphilis, 154 
Prurigo, eosinophilia in, 65 



Psoriasis, eosinophilia in, 65 
Puerperium, anemia in, 79 
Pulmonary tuberculosis, blood findings 
in, 86 
sputum in, 370 
Punctate basophilia of Grawitz, 53 
"Puncture headache," 372 
Purdy's method for determining albumin 
in urine, 242 
for quantitative determination 
of glucose in urine, 247 
Purpura hemorrhagica, prothrombin con- 
tent of blood in, 72 
Pus, 382. See Exudates, purulent, 
casts in urine, 227 
cells, 55 

in feces, 312, 318 

polymorphonuclear neutrophilic leu- 
kocytes in, 386 
in urine, 218, 229 
Pyelitis, laboratory findings in, 280, 282 
of pregnancy, blood cultures in, 103 
Pyuria, 229 



Rabbit, bleeding from ear, 133 

from femoral artery, 135 
from neck, 136 
intraperitoneal injections of, 131 
intravenous injections of, 131 
Rabies, examination of brain in, 400 
Reaction of media, 415 
Red blood cells. See Erythrocytes. 

volume of, 42 
Regan, J. C, reference to, 372 
Rehfuss tube, 290 
Reichmann's disease. See Gastrosuc- 

corrhea. 
Relapsing fever, 101 
Renal albuminuria, 222 

function, 237, 268, 271, 272, 273 
Rheumatism, acute articular, anemia in, 
79 
leukocytosis in, 65 
Ribierre, reference to, 72 
"Rice-bodies" in sputum, 356 
"Rice-water stools," 322 
Rickets, lymphocytosis in, 66 
Rieder's cells, 59 
Riegel's test-meal, 287 
Riesman, D., reference to, 383 
Ring-bodies of Cabot, 53 
Ringworm, 394 

Rivas and Smith, A. J., reference to, 100 
Roberts, reference to, 315 
Roberts' test for blood in urine, 237 
Robertson's cooked-meat medium, 429 
Robinson, A., reference to, 303 
Rose's method for determining pepsin, 

300 
Romanowsky group of stains, 47 
Ross-Jones test, 375 
Ross-Ruge method for concentrating 
blood for detecting malaria organisms, 
92 



INDEX 



479 



Rothera's test for acetone in urine, 234 
Round- worm infestations, eosinophilia 

in, 65 
Rous and Turner, method for blood 

matching, 114 
Russell's double-sugar agar, 428 



S 



Sabouraud, reference to, 395 

Safranin, 437 

Sahli's desmoid test, 304 

hemoglobinometer, 19 
Sarcinse in gastric contents, 298 
Scarlet fever, Dohle inclusion bodies in, 
90 
eosinophilia in, 65 
leukocytosis in, 65 
Schaudinn, reference to, 398 
Schistosomum hematobium, 331 
japonicum, 331 
Mansoni, 331 
Schmidt and Strasburger's test diet, 321 
Schwartz, reference to, 159 
Scorbutus, anemia in, 79 
Salzer's test-meal, 288 
"Seat-worms," 340. See Oxyuris ver- 

micularis. 
Secondary anemia, volume index in, 43 
Sediment in urine, 230 
Sellards, reference to, 198 
Sellards' test for acidosis by determina- 
tion of tolerance to sodium 
bicarbonate, 276 
reaction of protein-free blood 
serum to phenolphthalein, 
207 
Selling, reference to, 80 
Semen, examination of, 399, 400 
Sepsis, Dohle inclusion bodies in, 90 
Septicemia, diagnosis of, 86 
Serology, apparatus for, 447 
Serous cavities, tuberculosis of, 385 
Serum, spermatozoa in, 399 
Shawan, reference to, 112 
"Shreds" in urine, 218, 393 
Simon, reference to, 64, 241 
Skin, diseases of, eosinophilia in, 65 
grafting, blood grouping in, 112 
infections of, 393 
"Sleeping sickness," 99 
Slide-rule, use of, 274 
Smallpox, leukocytosis in, 65 
Smith, A. J., reference to, 343 
Smith and MacNeal, reference to, 121 
Smithies, reference to, 303 
Sodium carbonate solution, preparation 
of, 434 
hydrate solution, preparation of, 434 
Solis-Cohen, reference to, 86 
Solis-Cohen and Strickler, reference to, 

90 
Solutions, normal, preparation of, 432 
oxalic acid, 433 



Solutions, normal, preparation of, 
sodium carbonate, 
434 
hydrate, 434 
Sondern, reference to, 64 
Sondern's hypotheses, 87 
Spectroscope, 238 
Spectroscopic tests for hemoglobin and 

derivatives, 237 
Spermatozoa in semen, 399 
Spinal fluid, bacteriological examination 
of, 379 
influenza infection, 380 
meningococcus infec- 
tion, 380 
pneumococcus infec- 
tion, 380 
tubercle infection, 381 
cell-count of, significance of, 382 
characteristic findings in mor- 
bid conditions, 382 
chemical examination of, 374 
colloidal-gold reaction in, 375 
cytology of, 374 
differential count of cells in, 374 
examination of, 371 
routine, 373 

technic of spinal puncture 
371 
globulin content of, significance 

of, 382 
gross appearance of, 373 
significance of findings, 381 
Wassermann reaction with, 151. 
379 
significance of, 382 
puncture, technic of, 371 
Spirals, Curschmann's, in sputum, 351 

352 
Spirocheta berbera, 101 

buccalis in mouth and throat, 389 
dentium in mouth and throat, 389 
Duttoni, 101 

pallida, 396. See Treponema pal- 
lida. 
Spironema pallida, 396. See Treponema 

pallida. 
Spleen, enlargement of, accompanying 

anemia, 81 
Splenic anemia, erythrocytes in, resist- 
ance of, 73 
lymphocytosis in, 66 
Spores, stans for, 438 
Sputum, actinomycosis in, 354 
in acute bronchitis, 369 

lobar pneumonia, 370 
antiformin method of examining, 357 
Bacillus mucosus capsulatus in, 369 

smegmse in, 369 
blastomycetes in, 353 
in bronchial asthma, 370 
casts, fibrinous in, 350 
cholesterol crystals in, 353 
in chronic passive congestion of 
lung, 370 



480 



INDEX 



Sputum, concretions in, 351 
crystals in, 352 

cholesterol, 353 
fatty acid, 353 
hematoidin, 353 
Curschmann's spirals in, 351, 352 
Ditt rich's plugs in, 351 
clastic fibers, 352 
examination of, 348 

method of procedure in, 34S 
fatty acid crystals in, 353 
in fibrinous bronchitis, 370 
in gangrene of lung, 370 
gross appearance of, 349 
amount, 349 
color, 349 
consistency, 349 
description, 349 
odor, 349 
"heart-failure cells," 351 
hematoidin crystals in, 353 
hypomycetes in, 353 

cultivation of, 359 
"impression method" of making 

preparations of, 355 
influenza bacilli in, 359 
in malignant disease of lung, 370 
Micrococcus catarrhalis in, 367 
microscopic examination, 351 
monilia in, 353 
Much's granules in, 357 
necrotic tissue in, 351 
obtaining specimens of, 348 
parasites in, 351, 355 
pneumococcus in, 300 

cultivation of, 361, 362 
morphology of, 360 
typing of, 362 

agglutination method, 365 
precipitin method, 366 
in pulmonary tuberculosis, 370 
stained specimens of, 355 
staphylococci in, 367 
streptococci in, 367 
tubercle bacilli in, 356 

cultivation of, 358 
Staining methods, 435 
Stains for capsules, 438 
for flagella, 439 
preparation of. 435 
saturated solutions of, 435 
for spores, 438 
use of, 435 
"Stair-ease retention," 196 
Staphylococci, description of, 387 
Staphylococcus, infection with, blood 
cultures in, 104 
leukocytosis in, 65 
in mouth and throat, 389 
in spinal fluid, 381 
in sputum, 367 
Starvation, acidosis in, 23(3 
Stengel, reference to, 64 
Sterilizing, hot-air, 422 
steam, 422 



Stiles, reference to, 343 

Stitt, reference to, 99, 311, 331, 332, 326, 

337 
Stiffs apparatus for venipuncture, 124 
Stober, reference to, 353 
Stoke's reagent, 238 
Stomach, absorptive power of, 304 

carcinoma of, gastric findings in, 305 
chronic catarrh of , gastric findings in, 

306 
contents. See Gastric contents, 
continuous hypersecretion of, gastric 

findings in, 305 
dilatation of, gastric findings in, 304 
neuroses of, gastric findings in, 306 
tube, contraindications to use of, 
289 
technic of use of, 288 
ulcer of, gastric findings in, 304 
Stools. See Feces. 
Strauss' separatory funnel, 294 
Streptococci, description of, 387 
Streptococcus in ear, 389 

infection with, anemia in, 80 
blood cultures in, 104, 105 
Dohle inclusion bodies in, 90 
leukocytosis in, 65 
in mouth and throat, 389 
in spinal fluid, 381 
in sputum, 367 
Strickler, reference to, 86 
Strongvloides intestinalis, 343 
Subacidity, 298 

Sugar of blood, determination of, 169,171 
in urine, qualitative tests for, 223, 
226 
quantitative tests for, 242, 244, 

245 
significance of, 225 
Sugar-free broth, 424 
Sulphates in urine, 251 
Sulphosalicylic acid test for albumin in 

urine, 220 
Suppurations, chronic, anemia in, 80 
Swab-tubes, 415 

Swift and Ellis, reference to, 374, 375 
Syphilis, anemia in, 80 

complement-fixation tests for, 118 
congenital, lymphocytosis in, 65 
cure of, serological criteria of, 154 
of mouth and throat, 390 
primary, Treponema pallidum in, 

153, '356. 
provocative test in, 154 
Wassermann reaction at different 
stages of, 153 
Syphilitic unit, determination of, 145 



Taenia canina, 337. See Dipylidium 
caninum. 
dentata, 334. See Taenia solium. 



INDEX 



481 



Taenia dimunata, 337. See Hymenolepis 
dimunata. 
echinococcus, 334 
humana armata, 334. See Taenia 

solium, 
lata, 338. See Dibothriocephalus 

latus. 
mediocanellata, 332. See Taenia 

saginata. 
nana, 335. See Hymenolepis nana, 
saginata, 332 
solium, 334 
Taenias, infestation with, eosinophilia in, 

65 
Tallquist's hemoglobinometer, 24 
Tapeworm, Russian, 338. See Dibo- 
thriocephalus latus. 
"unarmed," 332. See Taenia sagi- 
nata. 
Test-meals, types of, 287, 288 
Temperature, effect of, on erythrocyte 

count, 38 
Thayer and Tileston, reference to, 82 
Thermometer scales, conversion of, 

453 
Thoma ruling of hemocytometers, 32 
Thomas, B. A., method of determining 

renal function, 271 
Thomson's medium, 156 

for gonococci, 426 
Thoracentesis, 383 

Thorax, fluid of, examination of, 383 
"Thread-worms," 340. See Oxyuris ver- 

micularis. 
Three-glass test, 277 
Throat and mouth, exudates in, exami- 
nation of, 389 
Bacillus diphtherise in, 391 
Diplococcus pneumoniae in, 389 
Endamceba gingivalis in, 389 
Lepothrix buccalis in, 389 
Oidium albicans in, 389 
Spirocheta buccalis in, 389 

dentium in, 389 
staphylococcus in, 389 
streptococcus in, 389 
Treponema pallidum in, 390 
syphilis of, 390 
Thrombosis of superior mesenteric artery, 

324 
Thrush, 389 
Tick, argasine, 101 
Tinea barbae, 394 
circinata, 394 
sycosis, 394 
tonsurans, 394 
Todd, reference to, 64 
Toison's diluting fluid, 33 
Topfer's method, 297 

reagent, 293 
Torrey, reference to, 156 
Trachoma, bodies of, 389 
Transfusion of blood, blood grouping in, 

112 
Transitional leukocytes, 57, 6Q 



Transudates and exudates, examination 
of, 383 
bacteriological, 385 
cytological, 384 
Trematodes, 329 

Treponema pallidum, detection of, 396 
in mouth and throat, 390 
in primary syphilis, 153 
Trichina spiralis, 345. 
Trichinella spiralis, description of, 345 

in tissues, 325 
Trichinosis, eosinophilia in, 65, 345 
Trichocephalus dispar, 346. 

trichiurus, 346. 
Trichomonas hominis, 327 
intestinalis, 327 
vaginalis, 328 
Trichuris trichiura, 346 
Trousseau's test for bile pigments in 

urine, 233 
Trychophyton megalosporon, 394 

mierosporon, 394 
Trypanosoma gambiense, 99 

rhodesiense, 99 
Trypanosomiasis, 99 
Trypsin, tests for, 307 
Tsuchiya's method for determining albu- 
min in urine, 242 
Tubercle bacillus. See Bacillus tuber- 
culosis. 
in feces, 320 
in sputum, 356 
in urine, detection of, 276 
Tuberculin, injections of, eosinophilia 

following, 65 
Tuberculosis, anemia in, 80 
Arneth formula in, 90 
complement-fixation test in, 159 
Dohle inclusion bodies in, 90 
lymphocytosis in, 65 
of meninges, 381, 382 
pulmonary, blood findings in, 86 

sputum in, 370 
of serous cavities, 385, 386 
Tumor tissue in gastric contents, 303 
Tumors, malignant, eosinophilia in, 65 

leukocytosis in, 65 
Tiirck's irritation forms, 58 

ruling of hemocytometers, 32 
Two-glass test, 227 
Typhoid bacilli in feces, 321 

fever, agglutination tests in, 111 
anemia in, 79 
blood cultures in, 104, 106 
diagnosis of, 86 
feces in, 321, 322 
lymphocytosis in, 65 
platelet count in, 41 
Tyrosine, crystals of, in urine, 231 
in sputum, 352 



Uffelman's test for lactic acid, 294 
Ulcer, gastric, anemias in, 79 



4S2 



INDEX 



Ulcer, gastric gastric findings in, 304 

Ulcers, intestinal, feces in, 324 

Urate, ammonium, in urine, 232 

Urates in urine, 218, 230, 232 

Urea in urine, 255, 259, 265 

Urease, preparation of, 177 

Urethra, Diplococcus gonorrhea? in, 393 

Micrococcus catarrhalis in, 393 
Urethritis, laboratory findings in, 281, 
282 
posterior, eosinophilia in, 65 
Uric acid, crystals of, in urine, 231 

reagent of Folin and Denis, 191 
in urine, normal quantity of, 
263, 265 
quantitative determination 
of, 261 
Urine, acetone in, significance of, 235 
tests for, 234 

Gunning's, 234 
Legal's, 234 
Rothera's, 234 
albumin in, quantitative tests for, 
220 
heat and acetic acid, 

220 
Heller's 220 
magnesium sulphate, 

220 
nitric acid ring, 220 
sulphosalicylic acid, 
220 
quantitative tests for, Esbach's, 
242 
Purdv's 242 
Tsuchiya's, 242 
significance of, 222 
ammonia in, 259, 261, 266 
determination of, 259 

colorimetric method, 260 
Folin's alternate clinical 
method, 260 
method, 259 
normal quantity, 261 
ammonium urate in, deposit of, 232 
amount of, daily, 218 

in twenty-four hours, 265 
appearance of specimen of, 218 
bacteria in, 218, 230 
bacteriological examination of, 276 
tubercle bacilli, detection 
of, 276 
Bence-Jones protein in, 240 
beta-oxybutyric acid in, tests for, 

235 
bile acids in, 233 
pigments in 233 

foam test for, 233 
Gmelin's test for, 233 
significance of, 233 
Trousseau's test for, 233 
blood in, 229, 237 

significance of, 238 
tests for, 237 

benzidine reaction, 237 



Urine, Mood in, tests for, spectroscopic, 
237 
calcium carl innate in, deposit of, 232 
casts in, 227 
blood, 227 
epithelial, 227 
fatty, 227, 228 
fibrin, 228 • 
granular, 227 
hyaline, 227 
leukocyte, 227 
pus, 227 

significance of, 228 
waxy, 228 
chemical composition of, normal, 265 
chlorides in. 249, 265 

normal quantity, 251, 265 
qualitative tests for, 249 
quantitative tests for, Harvey's 
250 
Mohr's 249 
Volhard's250 
significance of findings, 251 
cloudy specimens of, causes of, 218 
creatine in, 264 

and creatinine in, 263, 264, 265 
determination of, Folin's 
method, 263 
determination of, 264 

Folin-Benedict method, 264 
creatinine in, normal quantity, 264 
crystals in, 230 

of ammonium-magnesium phos- 
phate in, 231 
of bilirubin in, 231 
of calcium oxalate in, 231 
of cystine in, 231 
of hematoidin in, 231 
of hippuric acid in, 231 
of leucine in, 231 
of tyrosin in, 231 
of uric acid in, 231 
cylindroids, 228 
diacetic acid in, significance of, 235 

tests for, Gerhardt's, 235 
diazo reaction in, 239 
epithelial cells in, 229 
erythrocytes in, 229 
examination of, 217 

bacteriological, 276 

on large scale, 431 

microscopic, 226 

order of procedure in, 218 

quantitative determinations, 

242 
routine, 217 

with relation to life insurance, 
281 
to morbid conditions, 278 
findings in morbid conditions, 278 
in cystitis, 281 
in diabetes insipidus, 
281 
mellitus, 281 
in nephritis, acute, 27!) 



INDEX 



483 



Urine, findings in morbid conditions, 
in nephritis, 
amyloid, 279 
chronic intersti- 
tial, 279 
parenchymatous, 
279 
in nephrolithiasis, 280 
in passive congestion 

of kidneys, 280 
in pyelitis, 280 
in urethritis, 281 
indican in, significance of, 237 
tests for, 236 

Obermayer's, 236 
leukocytes in, 229 
mercury in, detection of, 266 
mucous shreds in, 218 
threads in, 229 , 
■of multiple myeloma, Bence-Jones 

protein in, 241 
nitrogen in, determination of, 251 
colorimetric method, 254 
normal quantity of, 255, 265 
total 251, 265 
nucleoprotein in, 241 
phosphates in, 218, 230, 251, 

265 
plates of neutral calcium phosphate 

in, 231 
preservation of, 218 
protein bodies in, qualitative tests 
for, 220 
quantitative tests for, 242 
pus in, 218, 229 
sediment of, 230 
"shreds" in, 218, 393 
specific gravity of, 218 
sugar in, qualitative tests for, 223 
Benedict's 223 
Fehling's 223 
fermentation 224 
phenylhydrazine reaction, 
224 
quantitative tests for, Bene- 
dict's 244 
Fehling's, 243 
fermentation, 242 
Lohnstein's saccharo- 

meter, 243 
Myers' modification of 

Benedict's, 245 
polariscopic, 248 
Purdy's, 247 
significance of, 225 
sulphates in, 251, 265 
testing of, hydrometer for, 219 
three-glass test of, 277 
two-glass test of, 277 
urates in, 218, 230 
urea in, 255, 259, 265 

determination of, 255 

Marshall's method, 256 
Myers's colorimetric 
method, 256 



Urine, urea in, determination of, Van 
Slyke and Cullen's method, 
256 
normal quantity, 259 
uric acid in, 231, 233, 2S5 
determination of, 261 

Benedict and Hitchcock's 

method, 261 
Folin and Denis's method, 

modified, 261 
normal quantity, 263 
urobilin in, 239 
urobilinogen in, 239 
yeast cells in, 230 
Urobilin, in urine, significance of, 239 

test for, 239 
Urobilinogen in urine, 239 



Vaccines, autogenous, preparation of, 
449 
preservation of, 451 
standardization of, 449 

Hopkins's method, 450 
Wright's method, 449 
sterilization of, 450 
Vagina, Diplococcus gonorrhese in, 393 

exudates of, examination of, 392 
Van Giesen's stain, 400 
Van Slyke and Cullen, method for deter- 
mining carbon-dioxide com- 
bining power of blood, 199 
urea in urine, 256 
reference to, 177 
Van Slyke, reference to, 27 
j Van Slyke and Donleavy, reference to, 

213, 214 
Van Slyke's gastrometric method for de- 
termining hemoglobin, 20 
! Vaquez's disease, 38 
Venipuncture, 122, 126 
j Vincent's angina, 390 

organisms of, 390 

in soft chancre, 399 
| Vital staining, 69, 70 
i Vogel and Lee's method for detecting 

mercury in urine, 266 
Volume-index of Capps, 43 
Vomiting, persistent, effect of, on ery- 
throcyte count, 38 
Von Jaksch's diseases, 81 
Von Wedel, reference to, 161 



W 



Warfield, reference to, 67 
Walker, reference to, 327, 329 
Walker's index of resistance, 88 
Waring, reference to, 101 
Wassermann reaction, alcohol, effect of, 
on reaction, 128 
amboceptor, hemolytic, 129,131 



484 



INDEX 



Wassermann reaction, amboceptor, 
native anti-sheep, 149 
preparation of, 131 
titration of, 133, 136 
antigens, 120, 129, 141 
acetone-insoluble, 142 
cholesterolized alcohobc 

extracts, 141 
dilution of, 147 
Ecker and Sasano's, 142 
Noguchi's, 142 
syphibtic, 142 
titration of, 143 
Bauer reaction in, 149 
in cerebrospinal syphilis, 155 
comparison of methods of, 120 
complement, 129, 131 
dilution of, 141 
preparation of, 137 
titration of, 139 
complement-fixation of, 121 

temperature for, 121 
controls, 147 
general principle of, 118 
modifications of, 121 
preliminary set of reactions for, 

147 
quantities used, 121 
reading reactions, 150 
reagents, 129 
results, interpretation of, 152, 

156 
saline solution for, 129 
serum, inactivation of, 120, 128 
setting up reactions, 148 
sheep cells for, 129 
specimens, how secured, 122 

in children, 127 
with spinal fluid, 151, 155, 379 
Waxy casts in urine, 228 
Webb and Williams, reference to, 57 
Webster, reference to, 64 
Weights and measures, table of, 451 
Welch's capsule stain, 438 
West's tube, 392 
White blood cells. See Leukocytes. 



Widal and Ravaut, reference to, 385 
Widal reaction, interpretation of results 
of, 111 
technic of, 107 

macroscopic method, 107 
microscopic method, 107 
William's stain, 400 
Williamson, reference to, 27, 67 
Wilson and von Wedel, method for tuber- 
culosis complement-fixation test, 161 
Wilson's stain, preparation of, 440 
Wood, reference to, 64, 239 
Wolf and Junghaus test for dissolved 

albumin in gastric contents, 302 
Wright and Kinnicutt, findings on plate- 
let counts, 41 
method for counting platelets, 
39 
Wright's method for standardization of 
autogenous vaccines, 459 
stain, 47 

preparation of, 440 
for treponemata, 399 



Xanthochromia, 373 



Yaoita's method of search for parasitic 

ova, 325 
Yeast cells in gastric contents, 298 
in urine, 230 



Ziehl-Neelsen's carbol-fuchsin, 435 
method of staining tubercle bacillus, 
356 
Zinsser, reference to, 118 
Zinsser, Hopkins and Ottenburg, refer- 
ence to, 118 



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